Infect Immun, 1990 Sep, 58(9), 2815 - 20 Anti-idiotypic antibodies counteract the invasion inhibition capacity of antibodies to major epitopes of the Plasmodium falciparum antigen Pf155/RESA; Wahlin B et al.; Rabbits were immunized with the synthetic peptide EENVEHDA or (EENV)2, corresponding to a tandemly repeated sequence in the C-terminal part of the Plasmodium falciparum antigen Pf155/RESA, or with Escherichia coli-derived fusion proteins containing the corresponding repeats . For all sera, the capacity of the total immunoglobulin G fractions to inhibit P . falciparum merozoite invasion in vitro was similar and relatively low . Affinity purification of Pf155/RESA-specific antibodies on parasite-infected erythrocyte monolayers or on peptide columns increased the inhibitory capacity 50 to 5,000 times, whereas the immunofluorescence titers were increased only 10 times . The addition of small amounts of total immunoglobulin G to the affinity-purified antibodies gave a marked and dose-dependent reduction of the inhibitory capacity of the purified antibodies . However, this reduction was only seen in combinations where the immunoglobulin G fraction was from the same serum as the affinity-purified antibodies, suggesting that it was mediated by anti-idiotypic antibodies reacting with non-cross-reacting idiotopes of the invasion-inhibiting antibodies. J Virol, 1990 Sep, 64(9), 4321 - 8 Template switching by reverse transcriptase during DNA synthesis; Luo GX et al.; The ability of reverse transcriptase to make template switches during DNA synthesis is implicit in models of retrovirus genome replication, as well as in recombination and oncogene transduction . In order to understand such switching, we used in vitro reactions with purified nucleic acids and enzymes . The assay system involved the use of an end-labeled DNA primer so as to allow the quantitation of elongation on a donor template relative to the amount of elongation achieved by template switching (by means of sequence homology) when an acceptor template RNA was added . We examined several variables that affected the efficiency of the reaction: (i) the reaction time, (ii) the relative amounts of acceptor and donor template, (iii) the extent of sequence overlap between the donor and acceptor templates, and (iv) the presence or absence of RNase H activity associated with the reverse transcriptase . The basic reaction, with RNA templates and normal reverse transcriptase, yielded as much as 83% template switching . In the absence of RNase H, switching still occurred but the efficiency was lowered . Also, when the donor template was changed from RNA to DNA, there was still switching; not surprisingly, this was largely unaffected by the presence or absence of RNase H . Finally, we examined the action of the RNase H on RNA templates after primary transcription but prior to template switching . We found that in most cases, both ends of the original RNA template were able to maintain an association with the DNA product . This result was consistent with the work of others who have shown that RNase H acts as an endonuclease. Appl Microbiol Biotechnol, 1990 Sep, 33(6), 680 - 2 Cloning of a gene coding for phosphotransacetylase from Escherichia coli; Yamamoto-Otake H et al.; A phosphotransacetylase gene (pta) has been cloned from a genomic DNA library of Escherichia coli 1100, a derivative of strain K-12 . The phosphotransacetylase activities of pta+ plasmid-containing strains were amplified about 150-fold under control of the lac promoter . The molecular weight of the phosphotransacetylase was estimated to be about 81,000 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis . The pta gene was found to be downstream of ackA by a combination of restriction analysis and plasmid subcloning . It is located about 13 kb upstream of the purF-folC-hisT region of the chromosome. Appl Microbiol Biotechnol, 1990 Sep, 33(6), 619 - 23 Effect of free-cell growth parameters on oxygen concentration profiles in gel-immobilized recombinant Escherichia coli; Huang J et al.; Oxygen concentration profiles in immobilized recombinant cells were measured using a microsensor . Experimental results showed that the final depth of oxygen penetration in the carrageenan gel was always in the range 80-100 microns for the strains and media used, although profiles of oxygen concentration during the early stages of cell growth depended on the strain and nutrient medium used . Variations in the oxygen profiles corresponded to differences in kinetic parameters measured for the same strains and media in free-cell experiments. Appl Microbiol Biotechnol, 1990 Sep, 33(6), 611 - 8 Measurement of oxygen concentration gradients in gel-immobilized recombinant Escherichia coli; Hooijmans CM et al.; In this study, an oxygen microsensor was used to measure oxygen concentration profiles in carrageenan gel particles containing growing, immobilized Escherichia coli B (pTG201) . Profiles, which were measured at intervals during continuous culture of gel slabs and beads, became increasingly steep with time . The oxygen penetration depth in the gel decreased with time, eventually reaching a steady state value of approximately 100 microns for both gel beads and slabs . A reaction-diffusion model employing zero-order cell growth kinetics was found to provide an excellent fit to the experimental concentration data . Growth rates estimated from profiles obtained during the first few hours of culture were 0.24h-1 (gel slabs) and 0.18h-1 (beads), compared to a value of 0.30 h-1 measured in free-cell suspensions at 25 degrees C. Biotechnology (N Y), 1990 Sep, 8(9), 849 - 53 Expression of intracellular hemoglobin improves protein synthesis in oxygen-limited Escherichia coli; Khosla C et al.; We have previously cloned the Vitreoscilla hemoglobin gene (VHb) and expressed the protein in Escherichia coli in its active form . Under oxygen-limited conditions the presence of VHb improves protein synthesis as indicated by both total protein content and the activity of an enzyme expressed from a cloned gene present on a multicopy plasmid . Measurements of nitrogen utilization rates corroborate the observation of enhanced protein synthesis; however, the rates of carbon consumption and acid synthesis remain unchanged . This suggests that the net effect of VHb in E . coli is to improve the efficiency, rather than the kinetics, of oxygen-limited aerobic metabolism . We propose two possible models for the mechanism of action of VHb: the facilitated diffusion hypothesis and the intracellular redox effector hypothesis . These suggest other systems in which cloned VHb may enhance bioprocess productivity. Biotechnology (N Y), 1990 Sep, 8(9), 841 - 4 A new vector for cloning large eukaryotic DNA segments in Escherichia coli; Leonardo ED et al.; We investigated the relationship between plasmid size and electroporation efficiency in E . coli and found that even very large plasmids can be transfected efficiently . The efficiencies are well above the minimum required to construct representative libraries of complex eukaryotic genomes . To exploit this observation we constructed a novel mammalian-E . coli shuttle vector whose replication in E . coli is driven by the F sex factor episome origin . This new vector system should accept inserts well in excess of 100 kb, thus putting the cloning of mammalian genes by direct phenotypic complementation within reach. Biotechnology (N Y), 1990 Sep, 8(9), 833 - 9 Inheritance and expression of chimeric genes in the progeny of transgenic maize plants; Fromm ME et al.; We obtained transgenic maize plants by using high-velocity microprojectiles to transfer genes into embryongenic cells . Two selectable genes were used to confer resistance to either chlorsulfuron or phosphinothricin, and genes encoding either E . coli beta-glucuronidase or firefly luciferase were used as markers to provide convenient assays for transformation . When regenerated without selection, only two of the eight transformed embryogenic calli obtained produced transgenic maize plants . With selection, transgenic plants were obtained from three of the other eight calli . One of the two initial lines produced 15 fertile transgenic plants . The progeny of these plants contained and expressed the foreign genes . Luciferase expression could be visualized, in the presence of added luciferin, by overlaying leaf sections with color film. Biophys Chem, 1990 Aug 31, 37(1-3), 183 - 96 Amino acid substitutions which stabilize aspartate transcarbamoylase in the R state disrupt both homotropic and heterotropic effects; Newell JO et al.; We have used site-specific amino acid substitutions to investigate the linkage between the allosteric properties of arpartate transcarbamoylase and the global conformational transition exhibited by the enzyme upon binding active-site ligands . Two mutationally altered enzymes in which an amino acid substitution had been introduced at a single position in the catalytic polypeptide chain (Lys-164----Glu and Glu-239----Lys) and a third species harboring both of these substitutions (Lys-164:Glu-239----Glu:Lys) were constructed . Sedimentation velocity difference studies were performed in order to assess the effects of the amino acid substitutions on the quaternary structure of the holoenzyme in the absence and presence of various active-site ligands, including the bisubstrate analog, N-(phosphonacetyl)-L-aspartate (PALA), which has been shown previously to promote the allosteric transition . In the absence of ligand, two of the mutationally altered enzymes, Lys-164----Glu and Lys-164:Glu-239----Glu:Lys, existed in the R conformation, isomorphous with that of the PALA-liganded wild-type holoenzyme . These enzymes exhibited no conformational change upon binding PALA . The unliganded Glu-239----Lys enzyme had an average sedimentation coefficient intermediate between that of the unliganded and PALA-liganded states of the wild-type enzyme which could be accounted for in terms of a mixture of T- and R-state molecules . This mutant enzyme was converted to the fully swollen conformation upon binding PALA, phosphate or carbamoyl phosphate . The allosteric properties of the mutationally altered species were investigated by PALA-binding studies and by steady-state enzyme kinetics . In each case, the mutationally altered enzymes were devoid of both homotropic and heterotropic effects, supporting the premise that the allosteric properties of the wild-type enzyme are linked to a ligand-promoted change in quaternary structure. J Chromatogr, 1990 Aug 31, 515, 483 - 94 Isolation and characterization of recombinant eel growth hormone expressed in Escherichia coli; Sugimoto S et al.; To obtain information about the microheterogeneity of recombinant protein, recombinant eel growth hormone II (EGH) analogues expressed in Escherichia coli were isolated and characterized . The modification was classified into three types: monodeamidation of Asn, oxidation of Met and N-terminal formylation . Monodeamidated EGH was isolated by ion-exchange chromatography . The major deamidation site (Asn 147) was determined by peptide mapping using the substrate specificity of trypsin . Oxidized EGH and N-terminal-formylated EGH were isolated by reversed-phase high-performance liquid chromatography . Oxidized EGH was identified by amino acid composition analysis and N-terminal-formylated peptide by mass spectrometry. Science, 1990 Aug 31, 249(4972), 1044 - 6 Electrostatic and steric contributions to regulation at the active site of isocitrate dehydrogenase; Dean AM et al.; The isocitrate dehydrogenase of Escherichia coli is regulated by covalent modification at the active site rather than, as expected, at an allosteric site . As a means of evaluating the mechanism of regulation, the kinetics of the substrate, 2R,3S-isocitrate, and a substrate analog, 2R-malate, were compared for the native, phosphorylated, and mutant enzymes . Phosphorylation decreases activity by more than a factor of 10(6) for the true substrate, but causes minor changes in the activity of the substrate analog . The kinetic results indicate that electrostatic repulsion and steric hindrance between the phosphoryl moiety and the gamma carboxyl group of 2R,3S-isocitrate are the major causes of the inactivation, with a lesser contribution from the loss of a hydrogen bond. Science, 1990 Aug 31, 249(4972), 1012 - 6 Regulation of an enzyme by phosphorylation at the active site; Hurley JH et al.; The isocitrate dehydrogenase of Escherichia coli is an example of a ubiquitous class of enzymes that are regulated by covalent modification . In the three-dimensional structure of the enzyme-substrate complex, isocitrate forms a hydrogen bond with Ser113, the site of regulatory phosphorylation . The structures of Asp113 and Glu113 mutants, which mimic the inactivation of the enzyme by phosphorylation, show minimal conformational changes from wild type, as in the phosphorylated enzyme . Calculations based on observed structures suggest that the change in electrostatic potential when a negative charge is introduced either by phosporylation or site-directed mutagenesis is sufficient to inactivate the enzyme . Thus, direct interaction at a ligand binding site is an alternative mechanism to induced conformational changes from an allosteric site in the regulation of protein activity by phosphorylation. Biochem Biophys Res Commun, 1990 Aug 31, 171(1), 169 - 74 Bioluminescent immunoassay using a fusion protein of protein A and the photoprotein aequorin; Zenno S et al.; Aequorin is a photoprotein that emits light in the presence of Ca2+ ions . To develop a bioluminescent immunoassay based on the light emission property of aequorin, we have expressed the apoaequorin fusion protein with S . aureus protein A in E . coli by recombinant DNA techniques . The fusion protein expressed was purified by IgG-Sepharose affinity chromatography, gel filtration and HPLC procedures . The purified protein A-apoaequorin fusion protein has both the luminescent activity of aequorin and the IgG-binding ability of protein A . We compared results obtained using the protein A-aequorin fusion protein with those obtained using a protein A conjugated horseradish peroxidase based immunoassay, and found them to yield similar results. J Chromatogr, 1990 Aug 31, 515, 563 - 72 High-performance liquid chromatography of transfer ribonucleic acids on spherical hydroxyapatite beads . II . Effects of pH and sodium chloride on chromatography; Yamakawa Y et al.; The effects of pH and sodium chloride on the high-performance liquid chromatography of transfer ribonucleic acids (tRNAs) on spherical hydroxyapatite beads were investigated . Binding of tRNAs on the beads was strengthened by increasing the pH of the mobile phase (phosphate buffer, pH 6.4-8.0), a phenomenon which is opposite to the retention of proteins on the beads . By using phosphate buffer of pH 8.0 instead of pH 6.8, the resolution of tRNAs was improved significantly; as many as ten times more theoretical plates were calculated with the use of the former buffer . Addition of sodium chloride to the phosphate buffer has a bifunctional effect on the retention of tRNAs: elution of tRNA(f-Met) and tRNA(Val) was retarded whereas that of tRNA(Phe) was facilitated. Biochem Biophys Res Commun, 1990 Aug 31, 171(1), 319 - 24 Properties of the exchange rate of guanine nucleotides to the novel rap-2B protein; Molina y Vedia L et al.; Rap-2B is a novel ras-related protein that is 89% identical to rap-2 at the amino acid level . Based on its amino acid sequence, it is anticipated that rap-2B binds guanine nucleotides . Here we show that purified, bacterially expressed rap-2B does bind both GTP and GDP in a Mg2(+)-dependent fashion . The relative affinity of rap-2B for GTP is higher than that for GDP, both at low and high concentrations of Mg2+ . This contrasts with N-ras p21 and could be of functional significance . Moreover, a polyclonal antiserum was raised against the recombinant rap-2B protein purified from E . coli lysates . This antiserum recognized a major protein of Mr approximately 21000 on Western blots of platelet membrane proteins, and immunoprecipitates rap-2B complexed with GTP or GDP. Biochem Biophys Res Commun, 1990 Aug 31, 171(1), 280 - 6 Cooperative mechanosensitive ion channels in Escherichia coli; Szabo I et al.; A patch-clamp investigation was carried out on giant Escherichia coli spheroplasts . The membrane exhibited stretch-induced as well as "spontaneous" activity, with similar characteristics, i.e., a large number of conductance values arising from the cooperative behavior of channels in functional clusters . It appears likely that the same molecular species are responsible for both stretch-induced and "spontaneous" current conduction; the channel multiplexes can either respond to membrane stretch or function in an activate state, presumably brought about by the previous application of the mechanical stimulus. Biochemistry, 1990 Aug 28, 29(34), 7890 - 4 Use of Mono Q high-resolution ion-exchange chromatography to obtain highly pure and active Escherichia coli RNA polymerase; Hager DA et al.; A method for the purification of highly pure and active Escherichia coli RNA polymerase holoenzyme is described . This method is simple, reproducible, and can be performed at room temperature . The procedure involves the high-performance liquid chromatography of a partially purified RNA polymerase sample on a Mono Q ion-exchange column . Under the conditions used, RNA polymerase holoenzyme is well separated from the core RNA polymerase and other impurities . The purified RNA polymerase contains virtually no impurities as judged by SDS-polyacrylamide gel electrophoresis . The purified RNA polymerase holoenzyme contains the sigma 70 subunit in stoichiometric amounts and is at least 90% active. Eur J Biochem, 1990 Aug 28, 192(1), 127 - 31 Expression of the chemically synthesized coding region for the cytotoxin alpha-sarcin in Escherichia coli using a secretion cloning vector; Henze PP et al.; The coding region for the cytotoxin alpha-sarcin from Aspergillus giganteus has been chemically synthesized by the ligation of 19 overlapping oligodeoxyribonucleotides . An Escherichia coli clone producing the cytotoxin was constructed by inserting the synthesized gene directly downstream to the region coding for the signal peptide of the OmpA protein (a major outer membrane protein of E . coli), using the secretion cloning vector pIN-III-OmpA2 . The enzyme encoded by the chemically synthesized gene expressed in E . coli displayed properties identical to those of native alpha-sarcin isolated from A . giganteus with respect to its chemistry, antigenicity and ribonucleolytic activity in qualitative assays. Biochim Biophys Acta, 1990 Aug 28, 1046(1), 97 - 105 Characterization of FadL-specific fatty acid binding in Escherichia coli; Black PN; The product of the fadL gene (FadL) is a central component of the long-chain fatty acid transport system of Escherichia coli . When fatty acid activation is blocked by a mutation in the structural gene for acyl CoA synthetase (fadD) transport is inhibited allowing a FadL-specific fatty acid binding activity to be measured . This binding activity was 4- to 6-fold greater in the fadL+ fadD strain LS6928 when compared to the delta fadLfadD strain LS6929 . With long-chain fatty acids, this binding activity was saturable and it was estimated that there were approx . 35,000 FadL-specific oleic acid binding sites per cell in the fadL+ strain LS6928 . The FadL-specific fatty acid binding affinity was highest for oleic acid (18:1) and palmitic acid (16:0) giving apparent KD values of 2.3.10(-7) M and 8.8.10(-7) M, respectively . FadL-specific binding affinity of myristic acid (14:0) was nearly an order of magnitude less and no FadL-specific binding of decanoic acid (10:0) could be measured . Two lines of evidence suggest that FadL-fatty acid binding occurs by a hydrophobic interaction: (1) There was a preference for the long-chain substrates oleic acid and palmitic acid; and (2) oleic acid binding activity was not significantly changed over the pH range 5.0 to 8.0 . The FadL-specific binding of oleic acid in the fadL+ strain LS6928 could be blocked by preincubation with antisera raised against purified FadL providing a clear correlation between the activity and identity of FadL . The binding activity associated with FadL was measured in vesicles of the outer membrane following passage over the hydrophobic resin Lipidex 1000 . The KD of oleic acid binding attributable to FadL in outer membranes vesicles (6.0.10(-7) M) was in close agreement with that determined in whole cells . Overall, these studies demonstrated that FadL binds long-chain fatty acids with a relatively high affinity prior to their transport across the outer membrane. Biochemistry, 1990 Aug 28, 29(34), 7882 - 90 RNA folding during transcription by Escherichia coli RNA polymerase analyzed by RNA self-cleavage; Monforte JA et al.; We have used a self-cleaving RNA molecule related to a subsequence of plant viroids (a "hammerhead") to study the length-dependent folding of RNA produced during transcription by Escherichia coli RNA polymerase . Transcript elongation is arrested at defined positions using chain-terminating ribonucleoside triphosphate analogues (3'-deoxyNTP's or 3'-O-methylNTP's) . When the transcript can form the "hammerhead" structure it self-cleaves to give a truncated product . The experiment yields an RNA sequencing ladder which terminates at the length at which cleavage becomes possible; the sequencing ladder is compared to those generated by using a noncleaving transcript or by using {alpha-thio}ATP in place of ATP . We have shown that 15-18 nucleotides (nt) of RNA past the cleavage point must be synthesized before the transcript can self-cleave within a ternary complex, whereas RNA freed from the complex by heating can cleave with only 3 or more nt present beyond the cleavage point . There are sequence-dependent as well as length-dependent effects . The results suggest that 12 +/- 1 nt are sequestered within the ternary complex and are consistent with the presence of a DNA-RNA hybrid within the transcription bubble, as proposed by others . The results indicate that the "hammerhead" structure does not disrupt the hybrid . It appears that the RNA beyond the hybrid is not restrained by interactions with the enzyme, since the last stem of the self-cleaving structure forms as soon as the RNA composing it emerges from the DNA-RNA hybrid . Self-cleaving of the transcript offers a simple structural probe for studying less well-characterized transcription complexes . The relevance of the results to models for transcription termination is discussed. Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 93 - 7 Escherichia coli 30S mutants lacking protein S20 are defective in translation initiation; Gotz F et al.; The 30S ribosomal subunits derived from Escherichia coli TA114, a a temperature-sensitive mutant lacking ribosomal protein S20, were shown to be defective in two ways: (a) they have a reduced capacity for association with the 50S ribosomal subunit which results in the impairment of most of the functions requiring a coordinated interaction between the two subunits; (b) they are defective in functions which do not require their interaction with the large subunit (i.e., the formation of ternary complexes with aminocyl-tRNAs and templates, including the formation of 30S initiation complexes with fMet-tRNA and mRNA) . The 30S (-S20) subunits seem to interact normally with both template and aminoacyl-tRNA individually, but appear to be impaired in the rate-limiting isomerization step leading to the formation of a codon-anticodon interaction in the P site. Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 84 - 92 The conformation of the initiator tRNA and of the 16S rRNA from Escherichia coli during the formation of the 30S initiation complex; Romby P et al.; The conformation of the E . coli initiator tRNA and of the 16S rRNA at different steps leading to the 30S.IF2.fMet-ARN(fMet).AUG.GTP complex has been investigated using several structure-specific probes . As compared to elongator tRNA, the initiator tRNA exhibits specific structural features in the anticodon arm, the T and D loops and the acceptor arm . Initiation factor 2 (IF2) interacts with the T-loop and the minor groove of the T stem of the RNA, and induces an increased flexibility in the anticodon arm . In the 30S initiation complex, additional protection is observed in the acceptor stem and in the anticodon arm of the tRNA . Within the 30S subunit, IF2 does not significantly shield defined portions of 16S rRNA, but induces both reduction and enhancement of reactivity scattered in the entire molecule . Most are constrained in a region corresponding to the cleft, the lateral protrusion and the part of the head facing the protrusion . All the reactivity changes induced by the binding of IF2 are still observed in the presence of the initiator tRNA and AUG message . The additional changes induced by the tRNA are mostly centered around the cleft-head-lateral protrusion region, near positions affected by IF2 binding. Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 8 - 13 The three-dimensional structure and function of Escherichia coli ribosomal RNA, as studied by cross-linking techniques; Brimacombe R et al.; A large number of intra-RNA and RNA-protein cross-link sites have been localized within the 23S RNA from E . coli 50 S ribosomal subunits . These sites, together with other data, are sufficient to constrain the secondary structure of the 23 S molecule into a compact three-dimensional shape . Some of the features of this structure are discussed, in particular, those relating to the orientation of tRNA on the 50 S subunit as studied by site-directed cross-linking techniques . A corresponding model for the 16S RNA within the 30 S subunit has already been described, and here a site-directed cross-linking approach is being used to determine the path followed through the subunit by messenger RNA. Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 38 - 44 Probing tRNA binding sites on the Escherichia coli 30 S ribosomal subunit with photoreactive analogs of the anticodon arm; Wower J et al.; Two analogs of the anticodon arm of yeast tRNAPhe (residues 28-43), in which G43 was replaced by the photoreactive nucleosides 2-azidoadenosine and 8-azidoadenosine, have been used to create 'zero-length' cross-links to ribosomal components at the peptidyl-tRNA binding site (P site) of 30 S subunits from the Escherichia coli ribosome . To prepare the analogs, 2-azidoadenosine and 8-azidoadenosine bisphosphates were first ligated to the 3' end of the anticodon-containing dodecanucleotide ACmUGmAAYA psi m5CUG from yeast tRNAPhe . The trinucleotide CAG was then joined to the 5' end of the resulting tridecanucleotide in a subsequent ligation . Both analogs bound to poly(U)-programmed 30 S subunits with affinities similar to that of the unmodified anticodon arm from yeast tRNAPhe . Irradiation of noncovalent complexes containing the photolabile analogs, poly(U) and 30 S ribosomal subunits with 300 nm light led to the covalent attachment of the anticodon arms to proteins S13 and S19 . Further analysis revealed that S13 accounted for about 80%, and S19 for about 20%, of the cross-linked material . Labeling of these two proteins with 'zero-length' cross-linking probes provides useful information about the location and orientation of P site-bound tRNA on the ribosome and permits a test of recently proposed models of the three-dimensional structure of the 30 S subunit. Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 343 - 50 The translational regulation of threonyl-tRNA synthetase . Functional relationship between the enzyme, the cognate tRNA and the ribosome; Moine H et al.; The E . coli threonyl-tRNA synthetase gene is negatively autoregulated at the translational level by a direct binding of the enzyme to the leader region of the thrS mRNA . This region folds in four well-defined domains . The enzyme binds to the leader at two major sites: the first is a stem-loop structure located in domain II upstream of the translational initiation site (domain I) which shares structural analogies with the anticodon arm of several tRNA(Thr) isoacceptors . The second site corresponds to a stable stem-loop structure located in domain IV . Both sites are separated by a large unpaired region (domain III) . In vivo and in vitro experiments show that the structural integrity of both sites is required for the regulatory process . The binding of the enzyme to its mRNA target site represses its translation by preventing the ribosome from binding to its attachment site . tRNA(Thr) suppresses this inhibitory effect by displacing the mRNA from the enzyme at both the upstream stem-loop structure and the tRNA-like anticodon arm. Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 34 - 7 Dissection of the 16S rRNA binding site for ribosomal protein S4; Sapag A et al.; The ribosomal protein S4 from Escherichia coli is essential for initiation of assembly of 30S ribosomal subunits . We have undertaken the identification of specific features required in the 16S rRNA for S4 recognition by synthesizing mutants bearing deletions within a 460 nucleotide region which contains the minimum S4 binding site . We made a set of large nested deletions in a subdomain of the molecule, as well as individual deletions of nine hairpins, and used a nitrocellulose filter binding assay to calculate association constants . Some small hairpins can be eliminated with only minor effects on S4 recognition, while three hairpins scattered throughout the domain (76-90, 376-389 and 456-476) are essential for specific interaction . The loop sequence of hairpin 456-476 is important for S4 binding, and may be directly recognized by the protein . Some of the essential features are in phylogenetically variable regions; consistent with this, Mycoplasma capricolum rRNA is only weakly recognized by S4, and no specific binding to Xenopus laevis rRNA can be detected. Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 337 - 42 Transcriptional organization of the S10, spc and alpha operons of Escherichia coli; Lindahl L et al.; We have investigated the transcription patterns at the inter-operon regions between the S10 and spc, and spc and alpha ribosomal protein operons of Escherichia coli . Newly synthesized transcripts were characterized by RNase T1 protection experiments, and accumulated transcripts were mapped with S1 nuclease . With both techniques we found that about 75% of the RNA polymerases transcribing the S10 operon terminated at the position of a typical rho-independent terminator . In contrast, most or all RNA polymerases transcribing the spc operon continued into the alpha operon . Nevertheless, we observed that about 30% of the transcripts of the alpha operon were initiated at the alpha operon promoter. Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 328 - 36 Translational control of ribosomal protein S15; Portier C et al.; The expression of ribosomal protein S15 is shown to be translationally and negatively autocontrolled using a fusion within a reporter gene . Isolation and characterization of several deregulated mutants indicate that the regulatory site (the translational operator site) overlaps the ribosome loading site of the S15 messenger . In this region, three domains, each exhibiting a stem-loop structure, were determined using chemical and enzymatic probes . The most downstream hairpin carries the Shine-Dalgarno sequence and the initiation codon . Genetic and structural data derived from mutants constructed by site-directed mutagenesis show that the operator is a dynamic structure, two domains of which can form a pseudoknot . Binding of S15 to these two domains suggests that the pseudoknot could be stabilized by S15 . A model is presented in which two alternative structures would explain the molecular basis of the S15 autocontrol. Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 317 - 22 Mapping of two promoters for elongation factor Tu within the structural gene for elongation factor G; Zengel JM et al.; The str operon of Escherichia coli contains the genes for ribosomal proteins S7 and S12 as well as elongation factors G and Tu (EF-G, EF-Tu) . We have previously reported that there is a secondary promoter for expression of EF-Tu mapping within the upstream fus gene encoding EF-G (Zengel, J.M . and Lindahl, L . (1982) Mol . Gen . Genet . 185, 487-492) and have identified several potential promoter sequences within fus (Zengel, J.M., Archer, R.H . and Lindahl, L . (1984) Nucleic Acids Res . 12, 2181-2192) . We have now further characterized this promoter activity . Measurements of transcription rates from various regions of the str operon in cells carrying the fus gene and the beginning of the tufA gene on a high copy number plasmid confirmed that transcription was initiated within a 600 bp EcoRI fragment in the distal portion of the fus gene . Furthermore, T1 nuclease mapping studies identified two 5' ends within this region, one about 400 bases upstream of tufA, the other about 270 bases upstream, suggesting that there are two tufA promoters within the fus gene . Both of these promoters are active in the intact chromosomal str operon. Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 302 - 6 Potential binding sites of the trans-activator FIS are present upstream of all rRNA operons and of many but not all tRNA operons; Verbeek H et al.; FIS, the Escherichia coli protein that stimulates the inversion of various DNA segments by binding to a recombinational enhancer, trans-activates a number of stable RNA operons and binds to the upstream activator sequence (UAS) of these operons (Nilsson et al . (1990) EMBO J . 9, 727) . In a search for potential FIS-binding sites we have compared UASs of other stable RNA operons with a consensus FIS-binding sequence, compiled by comparing recombinational enhancers . Such sites can thus be recognized upstream of all rRNA and 13 tRNA operons . Matching with the consensus sequence varied, suggesting that the affinity of FIS for the sites differed . Accordingly, FIS binding to an upstream sequence of the metY(nusA) operon was found to be weaker than that to the UAS of the thrU(tufB) operon . No FIS binding sites were found upstream three tRNA operons. Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 259 - 62 Third position base changes in codons 5' and 3' adjacent UGA codons affect UGA suppression in vivo; Buckingham RH et al.; The base sequence around nonsense codons affects the efficiency of nonsense codon suppression . Published data, comparing different nonsense sites in a mRNA, implicate the two bases downstream of the nonsense codon as major determinants of suppression efficiency . However, the results we report here indicate that the nature of the contiguous upstream codon can also affect nonsense suppression, as can the third (wobble) base of the contiguous downstream codon . These conclusions are drawn from experiments in which the two Ser codons UCU233 and UCG235 in a nonsense mutant form (UGA234) of the trpA gene in Escherichia coli have been replaced with other Ser codons by site-directed mutagenesis . Suppression of these trpA mutants has been studied in the presence of a UGA nonsense suppressor derived from glyT . We speculate that the non-site-specific effects of the two adjacent downstream bases may be largely at the level of the termination process, whereas more site-specific or codon-specific effects may operate primarily on the activity of the suppressor tRNA. Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 248 - 51 Growth rate dependence of global amino acid composition; Emilsson V et al.; The global amino acid composition of bacteria growing in different media has been studied . The data reveal significant changes in the amino acid composition in the growth rate range between 0.5 and 2.1 doublings per hour at 37 degrees C . The changes are consistent with a progressive simplification of the protein population and mRNA pools as the growth rates increase. Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 235 - 40 Characterization of protein synthesis factors from rabbit reticulocytes; Merrick WC et al.; As part of our efforts to characterize eukaryotic translation factors, we have sequenced a number of them chemically and inferred sequences from cDNA clones . To our surprise, there appears to be extensive identity of amino acid sequence in most factors characterized to date in that within mammalian species, usually greater than 99% identity is observed . Extreme examples are rabbit EF-1 alpha which is 100% identical to human EF-1 alpha and rabbit eIF-4AI and eIF-4AII which are 100% identical to mouse eIF-4AI and eIF-4AII for those amino acids sequenced (398/406 and 156/407, respectively) . An extended analysis has been made of EF-1 alpha which in rabbit has three different post-translational modifications, dimethyllysine, trimethyllysine and glycerylphosphorylethanolamine . A comparison of the primary structure of EF-1 alpha to E . coli EF-Tu indicates an overall sequence identity of 33% . However, within the amino terminal 180 amino acids (the GTP-binding domain), there are found regions of much greater identity (50/85 = 59%). Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 226 - 9 The interaction between aminoacyl-tRNA and the mutant elongation factors Tu AR and B0; Abrahams JP et al.; The binding of Tyr-{AEDANS-s2C}tRNA(Tyr) (Tyr-tRNA(Tyr) modified at the penultimate cytidine residue with a thio group at position 2 of the pyrimidine ring, to which an N-(acetylaminoethyl)-5-naphthylamine-1-sulfonic acid fluorescence group is attached) to mutant elongation factor (EF)-Tu species from E . coli, EF-TuAR (Ala-375----Thr) and EF-TuBO (Gly-222----Asp), both complexed to GTP, was investigated in absence of kirromycin by measuring the change in fluorescence of the modified tRNA induced by complex formation . The calculated dissociation constant in the case of EF-TuAR is about 4 nM and in the case of EF-TuB0, about 1 nM . These values are higher than that of wild-type EF-Tu, which was 0.24 nM measured with the same system . The affinity between either EF-TuB0.kirromycin.GDP or EF-TuB0.kirromycin.GTP on the one hand, and a mixture of aminoacyl-tRNAs on the other, was measured with zone-interference gel electrophoresis . The dissociation constants are 20 microM and 7 microM, respectively, a factor of about two higher than in the case of wild-type EF-Tu.kirromycin . These findings provide a clue for the observed increase in translational errors in strains carrying the mutations . Furthermore, the experiments with EF-TuB0.kirromycin deepen our understanding of the effects of the B0 mutation on the kirromycin phenotype of the mutant cells concerned. Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 222 - 5 Ternary complexes of Escherichia coli aminoacyl-tRNAs with the elongation factor Tu and GTP: thermodynamic and structural studies; Ott G et al.; The interaction of 18 different Escherichia coli aminoacyl-tRNA species with elongation factor Tu and GTP has been measured by a fluorescence titration assay under equilibrium conditions . The dissociation constants range from 1.9 +/- 0.2.10(-10) M up to 1020 +/- 250.10(-10) M depending on the nucleotide sequence, secondary structure and the chemical composition of the aminoacyl residue of the particular aminoacyl-tRNA . The 'aminoacyl domain' of tRNA consisting of the single stranded, four-nucleotide-long 3'-terminus, aminoacyl stem of seven base-pairs, T-stem and T-loop contains all elements necessary for binding EF-Tu.GTP . The efficiency of aminoacyl-tRNA interaction with EF-Tu.GTP is modulated by the sequence of this 'aminoacyl domain' and by natural modification of its nucleotide residues . An oligoribonucleotide resembling the aminoacyl stem of E.coli tRNA(Ala) and consisting of a four-membered 3'-end, a stem of seven base-pairs and a loop of six nucleotides was prepared by total chemical synthesis on a polymer support . It can be enzymatically aminoacylated by alanine but does not bind in its aminoacylated form to EF-Tu.GTP. Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 14 - 7 Is there a special function for U.G basepairs in ribosomal RNA? van Knippenberg PH, Formenoy LJ, Heus HA. U.G basepairs are well-established elements of RNA structure . The geometry of this pair is different, however, from classical Watson-Crick basepairs . This leads to an unusual stacking of the basepair: overlap with the basepair at the 5' side of the U (and the 3' side of the G) is strong (stacked) while it is weak with the basepair on the other side (destacked) . The closure of an RNA helix by a U.G pair will be energetically unfavourable when the U residue occupies the 5' end . In transfer RNA there is a strong selection against a 'destacked' U.G pair at helix ends . In the 16S rRNA model of Escherichia coli there are 72 U.G pairs of which 36 or 22 occupy a helix end, depending on how such an end is defined . There is a slight preference for 'stacked' U.G's in these positions . It is remarkable, however, that of 13 very conserved U.G pairs in the 16S (-like) rRNA, 7 occur at helix ends and that 5 of these have the 'destacked' configuration . It is suggested that these pairs, if they exist at all in a hydrogen-bounded form, are stabilized by co-axial stacking with other helices or by interaction with a protein. Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 110 - 8 Secondary structure at the 3' terminal region of RNA coliphages: comparison with tRNA; Adhin MR et al.; Secondary structure models for the 3' non-coding region of the four groups of coliphage RNA are proposed based on comparative sequence analysis and on previously published data on the sensitivity of nucleotides in MS2 RNA to chemical modification and enzymes . We report the following observations . (1) In contrast to the coding regions, the structure at the 3' terminus is characterized by stable regular helices . We note the occurrence of the loop sequences 5'-GUUCGC and 5'-CGAAAG, that are reported to confer exceptional stability to stem structures . These features are probably present to promote the segregation of mother and daughter strands during replication . (2) Comparison of homologous helices indicates that only those base pair substitutions are allowed that maintain the thermodynamic stability . (3) We have compared the structure of phage RNA with tRNA . Overall similarity is low, but one common element may exist . It is a quasi-continuous helix of 12 basepairs that could be the equivalent of the 12 basepair long coaxially stacked helix, formed by the T psi C arm and the aminoacyl acceptor arm in tRNA . As in tRNA, this structure element starts after the fourth nucleotide from the 3' end . (4) Phage RNA contains a large variable region of about 35 nucleotides bulging out from the quasi-continuous helix . We speculate that the variable loop in present-day tRNA could be the remnant of the variable region found in phage RNA . The variable region contains overlapping binding sites for the replicase enzyme and the maturation protein . This common binding site may serve as a switch from replication to packaging. Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 45 - 50 Interaction of tRNA with domain II of 23S rRNA; Hill WE et al.; The interaction of tRNA with domain II of 23S rRNA in E . coli ribosomes has been probed using short, complementary DNA oligodeoxyribonucleotides . Specifically, cDNA oligomers to the region 801-811 of the 23S rRNA were used to ascertain the interaction of this region with tRNA . It was found that when tRNA was bound to the P site, considerable competition occurred between tRNA and the cDNA oligomers which base paired with the nucleotides 807-811 . However, A-site bound tRNA neither displaced, nor was displaced, by cDNA oligomers to this region . Additionally, the binding of tRNA lacking the CACCA nucleotides on the 3' terminus was unaffected by the presence a cDNA oligomer complementary to nucleotides 803-811, indicating that the cDNA-tRNA competition was dependent on the 3' terminal nucleotides of tRNA. Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 193 - 6 Similarities and differences in the inhibition patterns of thiostrepton and viomycin: evidence for two functionally different populations of P sites when occupied with AcPhe-tRNA; Kutay UR et al.; According to the allosteric three-site model for the ribosomal elongation cycle, the reactions from the pre- to the post-translocational state and vice versa represent allosteric transitions which are catalyzed by elongation factor (EF)-G and EF-Tu, respectively . It has been shown recently that the non-related antibiotics thiostrepton and viomycin inhibit protein biosynthesis via a surprisingly similar mechanism . Both drugs primarily block the allosteric transitions in either direction (Hausner et al . (1988) J . Biol . Chem . 263, 13103-13111) . Here we show that the secondary effects of these antibiotics differ strikingly . When the P site of poly(U) programmed ribosomes is quantitatively filled with AcPhe-tRNA, thiostrepton stimulates the rate of the formation of AcPhe-puromycin 2-fold, whereas viomycin inhibits the puromycin reaction (up to 75% inhibition) . The thiostrepton-dependent stimulation is only observed when the drug is given before the P site is occupied; when thiostrepton is added after pre-filling the P site, the peptidyltransferase activity is not affected, in contrast to the translocation reaction, which is blocked irrespective of whether the drug is administered before or after tRNA is bound . The effects of both drugs became distinctly more pronounced when the P sites were saturated with AcPhe-tRNA as compared to half-saturated ribosomes . We conclude that roughly one half of the ribosomes, which first bind AcPhe-tRNA to the P site, carry this ligand in a different orientation to that of the second half of the ribosome population . These two populations probably reflect the P site in the pre- and post-translocational state, respectively. Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 174 - 8 The translation system of mammalian mitochondria; O'Brien TW et al.; Oligoribonucleotides and mRNA were used to define properties of the bovine mitoribosomal mRNA binding site . The RNA binding domain on the 28 S subunit spans approx . 80 nucleotides of the template, based on ribosome protection experiments, but the major interaction with the ribosome occurs over a 30 nucleotide stretch . The binding site for E . coli IF3 is conserved in bovine mitoribosomes, but mitochondrial factors appear essential for proper interaction of mRNA with mitoribosomes . The small subunit of bovine mitoribosomes contains a high-affinity binding site for guanyl nucleotides, further indication of specialized mechanisms for initiation complex formation and function of mammalian mitochondrial ribosomes. Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 307 - 11 Sequences upstream of the-35 hexamer of rrnB P1 affect promoter strength and upstream activation; Josaitis CA et al.; Transcription from Escherichia coli ribosomal RNA promoters is increased about 20-fold in vivo by a DNA sequence (the Upstream Activation Region, UAR) located upstream of the -35 conserved hexamer . The UAR stimulates transcription through two mechanisms: one which involves binding of the Fis protein to the UAR, and another mechanisms which functions in the absence of additional protein factors . We have previously constructed a collection of mutations in the region upstream of the -35 hexamer of rrnB P1 . Most of these mutations have either no effect on promoter activity or decrease activity 2-5-fold in vivo (Gaal, T., Barkei, J., Dickson, R.R., De Boer, H.A., De Haseth, P.L., Alavi, H . and Gourse, R.L.(1989) J . Bacteriol . 171, 4852-4861) . Two mutations leave both the -35 consensus hexamer and the Fis binding consensus sequence intact, yet have larger (14-50-fold) effects on transcription . One substitution just upstream of the -35 hexamer (a C to T change at position -37) primarily affects intrinsic promoter strength, leaving the UAR functional . On the other hand, a three base pair deletion (bases -38 through -40) severely reduces UAR-mediated activity . A substitution covering the three base pair deletion was constructed and found to be activated normally . UAR function appears dependent on its position relative to the RNA polymerase binding site, suggesting that a particular spatial geometry may be necessary for Fis-dependent and/or factor-independent activation to occur. Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 215 - 21 Mutagenesis of the NH2-terminal domain of elongation factor Tu; Gumusel F et al.; Mutagenesis was carried out in the N-terminal domain of elongation factor Tu (EF-Tu) to characterize the structure-function relationships of this model GTP binding protein with respect to stability, the interaction with GTP and GDP, and the catalytic activity . The substitutions were introduced in elements around the guanine nucleotide binding site or in the loops defining this site, in the intact molecule or in the isolated N-terminal domain (G domain) . The double substitution Val88----Asp and Leu121----Lys, two residues situated on two vicinal alpha-helices, influences the basic activities of the truncated factor to a limited extent, probably via long-range interactions, and induces a destabilisation of the G domain structure . The functional alterations brought about by substitutions on the consensus sequences 18-24 and 80-83 highlight the importance of these residues for the interaction with GTP/GDP and the GTPase activity . Mutations concerning residues interacting with the guanine base lead to proteins in large part insoluble and inactive . In one case, the mutated protein (EF-TuAsn135----Asp) inhibited the growth of the host cell . This demonstrates the crucial role of the base specificity for the active conformation of EF-Tu . The obtained results are discussed in the light of the three-dimensional structure of EF-Tu. Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 209 - 14 Three-dimensional models of the GDP and GTP forms of the guanine nucleotide domain of Escherichia coli elongation factor Tu; Jurnak F et al.; Three-dimensional models of the GDP and GTP forms of the guanine nucleotide domain of Escherichia coli elongation factor Tu have been derived from the atomic coordinates of the trypsin-modified form of EF-Tu-GDP and by comparison with the ras p21 structures . The significance of the differences in the guanine nucleotide binding sites of EF-Tu and ras p21 are discussed . Crystallization of the EF-Tu-GMPPNP complex is reported. Nucleic Acids Res, 1990 Aug 25, 18(16), 4883 - 90 Crosslinking of tRNA containing a long extra arm to elongation factor Tu by trans-diamminedichloroplatinum(II); Rasmussen NJ et al.; A tRNA containing a long extra arm, namely E . coli tRNA(Leu1) has been crosslinked to elongation factor Tu, with the crosslinking reagent trans-diamminedichloroplatinum(II) . The nucleotide involved in the crosslinking was identified to be a guanosine in the variable region at position 47F or 47G. Nucleic Acids Res, 1990 Aug 25, 18(16), 4677 - 82 The role of modified purine 64 in initiator/elongator discrimination of tRNA(iMet) from yeast and wheat germ; Kiesewetter S et al.; The role of 2'-ribosylated adenosine 64 in tRNA(iMet) from yeast in initiation/elongation discrimination was investigated . As measured by in vitro translation in rabbit reticulocyte lysate, the specific removal of the 2'-ribosylphosphate at adenosine 64 via periodate oxidation allows tRNA(iMet) to read internal AUG codons of the globine messenger RNA . Yeast Met-tRNA(iMet) lacking the modification of nucleoside 64 forms ternary complexes with GTP and elongation factor Tu from Escherichia coli . The lack of modification at position 64 does not prevent tRNA(iMet) from participating in the initiation process of in vitro protein synthesis . Wheat germ tRNA(iMet) has a 2'-ribosylated guanosine at position 64 . Removal of this modification from the wheat germ tRNA(iMet) enables it to read internal AUG codons of globine and tobacco mosaic virus messenger RNA in reticulocyte and wheat germ translation systems, respectively. Ned Tijdschr Geneeskd, 1990 Aug 25, 134(34), 1651 - 3 {The pneumoportogram, a rare finding in septicemia due to an intra-abdominal abscess}; Leenen LP et al.; A man aged 73 is described with radiologically revealed air in the portal vein, a peumoportogram, on the basis of intra-abdominal sepsis . Characteristic of this finding are the air shadows extending far into the periphery of the liver in contrast to an air cholangiogram . It is an ominous sign, mostly secondary to severe intra-abdominal pathology such as ischaemic bowel necrosis, peritonitis or abscesses . Treatment should be aimed at these causes . Prognosis depends on the underlying pathology. Nucleic Acids Res, 1990 Aug 25, 18(16), 4659 - 64 Cloning and nucleotide sequence of the genes coding for the Sau96I restriction and modification enzymes; Szilak L et al.; The genes coding for the GGNCC specific Sau96I restriction and modification enzymes were cloned and expressed in E . coli . The DNA sequence predicts a 430 amino acid protein (Mr: 49,252) for the methyltransferase and a 261 amino acid protein (Mr: 30,486) for the endonuclease . No protein sequence similarity was detected between the Sau96I methyltransferase and endonuclease . The methyltransferase contains the sequence elements characteristic for m5C-methyltransferases . In addition to this, M.Sau96I shows similarity, also in the variable region, with one m5C-methyltransferase (M.SinI) which has closely related recognition specificity (GGA/TCC) . M.Sau96I methylates the internal cytosine within the GGNCC recognition sequence . The Sau96I endonuclease appears to act as a monomer. J Biol Chem, 1990 Aug 25, 265(24), 14669 - 74 Contacts between the factor TUF and RPG sequences; Vignais ML et al.; The yeast TUF factor binds specifically to RPG-like sequences involved in multiple functions at enhancers, silencers, and telomeres . We have characterized the interaction of TUF with its optimal binding sequence, rpg-1 (1-ACACCCATACATTT-14), using a gel DNA-binding assay in combination with methylation protection and mutagenesis experiments . As many as 10 base pairs appear to be engaged in factor binding . Analysis of a collection of 30 different RPG mutants demonstrated the importance of 8 base pairs at position 2, 3, 4, 5, 6, 7, 10, and 12 and the critical role of the central GC pair at position 5 . Methylation protection data on four different natural sites confirmed a close contact at positions 4, 5, 6, and 10 and suggested additional contacts at base pairs 8, 12, and 13 . The derived consensus sequence was RCAAYCCRYNCAYY . A quantitative band shift analysis was used to determine the equilibrium dissociation constant for the complex of TUF and its optimal binding site rpg-1 . The specific dissociation constant (K8) was found to be 1.3 x 10(-11) M . The comparison of the K8 value with the dissociation constant obtained for nonspecific DNA sites (Kn8 = 8.7 x 10(-6) M) shows the high binding selectivity of TUF for its specific RPG target. J Biol Chem, 1990 Aug 25, 265(24), 14579 - 91 Identification of residues critical for the polymerase activity of the Klenow fragment of DNA polymerase I from Escherichia coli; Polesky AH et al.; The Klenow fragment structure, together with many biochemical experiments, has suggested a region of the protein that may contain the polymerase active site . We have changed 7 amino acid residues within this region by site-directed mutagenesis, yielding 12 mutant proteins which have been purified and analyzed in vitro . The results of steady-state kinetic determinations of Km(dNTP) and kcat for the polymerase reaction, together with measurements of DNA binding affinity, suggest strongly that this study has succeeded in targeting important active site residues . Moreover, the in vitro data allow dissection of the proposed active site region into two clusters of residues that are spatially, as well as functionally, fairly distinct . Mutations in Tyr766, Arg841, and Asn845 cause an increase in Km(dNTP), suggesting that contacts with the incoming dNTP are made in this region . Mutations in the second cluster of residues, Gln849, Arg668, and Asp882, cause a large decrease in kcat, suggesting a role for these residues in catalysis of the polymerase reaction . The DNA-binding properties of mutations at positions 849 and 668 may indicate that the catalytic role of these side chains is associated with their interaction with the DNA substrate . Screening of the mutations in vivo for the classical polA-defective phenotype (sensitivity to DNA damage) demonstrated that a genetic screen of this type may be a reasonable predictor or kcat or of DNA binding affinity in future mutational studies. J Biol Chem, 1990 Aug 25, 265(24), 14536 - 43 Molecular analysis of the Escherichia coli ferric enterobactin receptor FepA; Armstrong SK et al.; In Escherichia coli, the outer membrane protein FepA is a receptor for the siderophore complex ferric enterobactin and for colicins B and D . To identify protein domains important for FepA activity, the effects of deletion and linker insertion mutations on receptor structure and function were examined . In-frame internal deletion mutations removing sequences encoding up to 304 amino acid residues resulted in functionally defective FepA polypeptides, although most were translocated efficiently to the outer membrane . One exception, a derivative lacking 87 internal amino acid residues near the N terminus, showed an inability to transport ferric enterobactin but retained limited colicin receptor function . Analysis of cells carrying 3'-terminal fepA deletion mutations suggested that residues within the C terminus of FepA may be involved in secretion and proper translocation of the protein to the outer membrane . Introduction of the peptide Leu-Glu after FepA residues 55, 142, or 324 severely impaired receptor function for all three ligands, while the same insertion after residues 339 or 359 had virtually no detrimental effect on FepA function . Foreign peptides inserted after residues 204 or 635 restricted colicin B and D function only, leaving ferric enterobactin transport ability at near wild-type levels . The results presented in this study have identified key regions of FepA potentially involved in receptor function and demonstrate the presence of both shared and unique ligand-responsive domains. J Biol Chem, 1990 Aug 25, 265(24), 14327 - 34 Metal binding to DNA polymerase I, its large fragment, and two 3',5'-exonuclease mutants of the large fragment; Mullen GP et al.; DNA polymerase I (Pol I) is an enzyme of DNA replication and repair containing three active sites, each requiring divalent metal ions such as Mg2+ or Mn2+ for activity . As determined by EPR and by 1/T1 measurements of water protons, whole Pol I binds Mn2+ at one tight site (KD = 2.5 microM) and approximately 20 weak sites (KD = 600 microM) . All bound metal ions retain one or more water ligands as reflected in enhanced paramagnetic effects of Mn2+ on 1/T1 of water protons . The cloned large fragment of Pol I, which lacks the 5',3'-exonuclease domain, retains the tight metal binding site with little or no change in its affinity for Mn2+, but has lost approximately 12 weak sites (n = 8, KD = 1000 microM) . The presence of stoichiometric TMP creates a second tight Mn2+ binding site or tightens a weak site 100-fold . dGTP together with TMP creates a third tight Mn2+ binding site or tightens a weak site 166-fold . The D424A (the Asp424 to Ala) 3',5'-exonuclease deficient mutant of the large fragment retains a weakened tight site (KD = 68 microM) and has lost one weak site (n = 7, KD = 3500 microM) in comparison with the wild-type large fragment, and no effect of TMP on metal binding is detected . The D355A, E357A (the Asp355 to Ala, Glu357 to Ala double mutant of the large fragment of Pol I) 3',5'-exonuclease-deficient double mutant has lost the tight metal binding site and four weak metal binding sites . The binding of dGTP to the polymerase active site of the D355A,E357A double mutant creates one tight Mn2+ binding site with a dissociation constant (KD = 3.6 microM), comparable with that found on the wild-type enzyme, which retains one fast exchanging water ligand . Mg2+ competes at this site with a KD of 100 microM . It is concluded that the single tightly bound Mn2+ on Pol I and a weakly bound Mn2+ which is tightened 100-fold by TMP are at the 3',5'-exonuclease active site and are essential for 3',5'-exonuclease activity, but not for polymerase activity . Additional weak Mn2+ binding sites are detected on the 3',5'-exonuclease domain, which may be activating, and on the polymerase domain, which may be inhibitory . The essential divalent metal activator of the polymerase reaction requires the presence of the dNTP substrate for tight metal binding indicating that the bound substrate coordinates the metal.(ABSTRACT TRUNCATED AT 400 WORDS) J Biol Chem, 1990 Aug 25, 265(24), 14227 - 33 Serine hydroxymethyltransferase catalyzes the hydrolysis of 5,10-methenyltetrahydrofolate to 5-formyltetrahydrofolate; Stover P et al.; The combined activities of rabbit liver cytosolic serine hydroxymethyltransferase and C1-tetrahydrofolate synthase convert tetrahydrofolate and formate to 5-formyltetrahydrofolate . In this reaction C1-tetrahydrofolate synthase converts tetrahydrofolate and formate to 5,10-methenyltetrahydrofolate, which is hydrolyzed to 5-formyltetrahydrofolate by a serine hydroxymethyltransferase-glycine complex . Serine hydroxymethyltransferase, in the presence of glycine, catalyzes the conversion of chemically synthesized 5,10-methenyltetrahydrofolate to 5-formyltetrahydrofolate with biphasic kinetics . There is a rapid burst of product that has a half-life of formation of 0.4 s followed by a slower phase with a completion time of about 1 h . The substrate for the burst phase of the reaction was shown not to be 5,10-methenyltetrahydrofolate but rather a one-carbon derivative of tetrahydrofolate which exists in the presence of 5,10-methenyltetrahydrofolate . This derivative is stable at pH 7 and is not an intermediate in the hydrolysis of 5,10-methenyltetrahydrofolate to 10-formyltetrahydrofolate by C1-tetrahydrofolate synthase . Cytosolic serine hydroxymethyltransferase catalyzes the hydrolysis of 5,10-methenyltetrahydrofolate pentaglutamate to 5-formyltetrahydrofolate pentaglutamate 15-fold faster than the hydrolysis of the monoglutamate derivative . The pentaglutamate derivative of 5-formyltetrahydrofolate binds tightly to serine hydroxymethyltransferase and dissociates slowly with a half-life of 16 s . Both rabbit liver mitochondrial and Escherichia coli serine hydroxymethyltransferase catalyze the conversion of 5,10-methenyltetrahydrofolate to 5-formyltetrahydrofolate at rates similar to those observed for the cytosolic enzyme . Evidence that this reaction accounts for the in vivo presence of 5-formyltetrahydrofolate is suggested by the observation that mutant strains of E . coli, which lack serine hydroxymethyltransferase activity, do not contain 5-formyltetrahydrofolate, but both these cells, containing an overproducing plasmid of serine hydroxymethyltransferase, and wild-type cells do have measurable amounts of this form of the coenzyme. Nucleic Acids Res, 1990 Aug 25, 18(16), 4867 - 76 Further biochemical characterization of wheat DNA primase: possible functional implication of copurification with DNA polymerase A; Laquel P et al.; DNA primase has been partially purified from wheat germ . This enzyme, like DNA primases characterized from many procaryotic and eucaryotic sources, catalyses the synthesis of primers involved in DNA replication . However, the wheat enzyme differs from animal DNA primase in that it is found partially associated with a DNA polymerase which differs greatly from DNA polymerase alpha . Moreover, the only wheat DNA polymerase able to initiate on a natural or synthetic RNA primer is DNA polymerase A . In this report we describe in greater detail the chromatographic behaviour of wheat DNA primase and its copurification with DNA polymerase A . Some biochemical properties of wheat DNA primase such as pH optimum, Mn + 2 or Mg + 2 optima, and temperature optimum have been determined . The enzyme is strongly inhibited by KCI, cordycepine triphosphate and dATP, and to a lesser extent by cAMP and formycine triphosphate . The primase product reaction is resistant to DNAse digestion and sensitive to RNAse digestion . Primase catalyses primer synthesis on M13 ssDNA as template allowing E.coli DNA polymerase I to replicate the primed M13 single-stranded DNA leading to double-stranded M13 DNA (RF) . M13 replication experiments were performed with wheat DNA polymerases A, B, CI and CII purified in our laboratory . Only DNA polymerase A is able to recognize RNA-primed M13 ssDNA. J Biol Chem, 1990 Aug 25, 265(24), 14195 - 201 Structure-function studies of adenine nucleotide transport in mitochondria . I . Construction and genetic analysis of yeast mutants encoding the ADP/ATP carrier protein of mitochondria; Lawson JE et al.; The gene encoding the major ADP/ATP carrier in yeast AAC2 (pet9; Lawson, J., and Douglas, M . (1988) J . Biol . Chem . 263, 14812-14818) has been disrupted (delta AAC2) by itself and in combination with a disruption of a second translocator gene AAC1 (delta AAC1) . Disruption of AAC2 like the pet9 mutation renders yeast unable to grow on a nonfermentable carbon source . The AAC1 AAC2 double disruption exhibits a phenotype identical to the AAC2 . This provides the host strain for the analysis of point mutations in the AAC protein . We have initiated this structure-function analysis by characterizing and confirming that the pet9 mutation is a G to A transition resulting in an arginine to histidine change at position 96 . Site-directed replacements at Arg96 confirm its essential function for growth on a nonfermentable carbon source . These data also suggest that in the absence of functional AAC1 and AAC2 gene products, adenine nucleotide transport across the mitochondrial inner membrane must occur by an as yet unidentified translocator or translocation mechanism or that within these cells separate intra- and extramitochondrial adenine nucleotide pools can exist to support growth. J Biol Chem, 1990 Aug 25, 265(24), 14632 - 7 Rat liver mitochondrial ATP synthase . Effects of mutations in the glycine-rich region of a beta subunit peptide on its interaction with adenine nucleotides; Garboczi DN et al.; The beta subunit of the rat liver mitochondrial ATP synthase contains a glycine-rich amino acid sequence implicated in binding nucleotides by its similarity to a sequence found in many other nucleotide-binding proteins . A C-terminal three-quarter-length rat liver beta subunit fragment (Glu122 through Ser479), containing this homology region, interacts with adenine nucleotides (Garboczi, D.N., Hullihen, J.H., and Pedersen, P.L . (1988) J . Biol . Chem . 263, 15694-15698) . Here we directly test the involvement of the glycine-rich region in nucleotide binding by altering its amino acid sequence through mutation or deletion . Twenty-one mutations within the glycine-rich region of the beta subunit cDNA were randomly generated . Wild-type and mutant beta subunit proteins were purified from overexpressing Escherichia coli strains . The mutant proteins were screened for changes in their interaction with 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP), a fluorescent nucleotide analog . Only one mutant protein bearing two amino acid changes (Gly153----Val, Gly156----Arg) exhibited a fluorescence enhancement higher than that of the wild-type "control." Further analysis of this protein revealed a lower affinity for TNP-ATP (Kd = 10 microM) compared with wild type (Kd = 5 microM) . In addition, a mutant from which amino acids Gly149-Lys214 had been deleted was prepared . This mutant protein, which lacks the entire glycine-rich region, also displayed a marked reduction in affinity for TNP-ATP (Kd greater than 60 microM) . Prior addition of 0.5 mM ATP significantly reduced the binding of TNP-ATP to both the double and deletion mutants . The altered interaction of nucleotide with both glycine-rich region mutants points to the involvement of this region in the binding site . Further, this work shows that a beta subunit protein that lacks the glycine-rich homology region can still interact with nucleotide, indicating that one or more additional regions of this subunit contribute to the nucleotide binding site. J Biol Chem, 1990 Aug 25, 265(24), 14606 - 11 Functional properties of the recombinant kringle-2 domain of tissue plasminogen activator produced in Escherichia coli; Wilhelm OG et al.; The kringle-2 domain (residues 176-262) of tissue-type plasminogen activator (t-PA) was cloned and expressed in Escherichia coli . The recombinant peptide, which concentrated in cytoplasmic inclusion bodies, was isolated, solubilized, chemically refolded, and purified by affinity chromatography on lysine-Sepharose to apparent homogeneity . {35S}Cysteine-methionine-labeled polypeptide was used to study the interactions of kringle-2 with lysine, fibrin, and plasminogen activator inhibitor-1 . The kringle-2 domain bound to lysine-Sepharose and to preformed fibrin with a Kd = 104 +/- 6.2 microM (0.86 +/- 0.012 binding site) and a Kd = 4.2 +/- 1.05 microM (0.80 +/- 0.081 binding site), respectively . Competition experiments and direct binding studies showed that the kringle-2 domain is required for the formation of the ternary t-PA-plasminogen-intact fibrin complex and that the association between the t-PA kringle-2 domain and fibrin does not require plasmin degradation of fibrin and exposure of new COOH-terminal lysine residues . We also observed that kringle-2 forms a complex with highly purified guanidine-activated plasminogen activator inhibitor-1, dissociable by 0.2 M epsilon-aminocaproic acid . The kringle-2 polypeptide significantly inhibited tissue plasminogen activator/plasminogen activator inhibitor-1 interaction . The kringle-2 domain bound to plasminogen activator inhibitor-1 in a specific and saturable manner with a Kd = 0.51 +/- 0.055 microM (0.35 +/- 0.026 binding site) . Therefore, the t-PA kringle-2 domain is important for the interaction of t-PA not only with fibrin, but also with plasminogen activator inhibitor-1 and thus represents a key structure in the regulation of fibrinolysis. Nucleic Acids Res, 1990 Aug 25, 18(16), 4695 - 701 Identification and characterization of a Dictyostelium discoideum ribosomal protein gene; Szymkowski DE et al.; We have identified a developmentally repressed large-subunit ribosomal protein gene of Dictyostelium discoideum based on sequence similarity to other ribosomal proteins . Protein rpl7 is homologous to large subunit ribosomal proteins from the rat and possibly to Mycoplasma capricolum and Escherichia coli, but is not similar to three sequenced ribosomal proteins in Dictyostelium . The rpl7 gene is present at one copy per genome, as are six other cloned Dictyostelium ribosomal proteins . Restriction fragment length polymorphisms exist for ribosomal protein genes rpl7, rp1024, and rp110 in strain HU182; most Dictyostelium ribosomal protein genes examined are linked no closer than 30-100 kb to each other in the genome . Dictyostelium ribosomal proteins are known to be developmentally regulated, and levels of rpl7 transcript gradually decrease during the 24-hour development cycle . This drop correlates with that of rp1024, indicating these and other ribosomal protein genes may be coordinately regulated . To determine the cellular location of the protein, we raised antibodies to an rpl7-derived branched synthetic peptide . These antibodies cross-reacted with one protein of the expected size in a ribosomal protein fraction of Dictyostelium, indicating that the product of gene rpl7 is localized in the ribosome. Science, 1990 Aug 24, 249(4971), 884 - 91 De novo design, expression, and characterization of Felix: a four-helix bundle protein of native-like sequence; Hecht MH et al.; The protein Felix was designed de novo to fold into an antiparallel four-helix bundle of specific topology . Its sequence of 79 amino acid residues is not homologous to any known protein sequence, but is "native-like" in that it is nonrepetitive and contains 19 of the 20 naturally occurring amino acids . Felix has been expressed from a synthetic gene cloned in Escherichia coli, and the protein has been purified to homogeneity . Physical characterization of the purified protein indicates that Felix (i) is monomeric in solution, (ii) is predominantly alpha-helical, (iii) contains a designed intramolecular disulfide bond linking the first and fourth helices, and (iv) buries its single tryptophan in an apolar environment and probably in close proximity with the disulfide bond . These physical properties rule out several alternative structures and indicate that Felix indeed folds into approximately the designed three-dimensional structure. Biochim Biophys Acta, 1990 Aug 24, 1027(2), 155 - 62 Lipid and peptide specificities in signal peptide--lipid interactions in model membranes; Demel RA et al.; The present data show the critical importance of the anionic lipid content in monomolecular layers for the interaction with PhoE signal peptide . At 37 degrees C and 100 mM NaCl the interaction is maximal at 30-40 mol% anionic lipid . The results correlate with the reduced translocation competence of Escherichia coli strain HD3122, which has a much lower anionic lipid content as compared to the wild-type strain SD12 (De Vrije et al . (1988) Nature 334, 173-175) . PhoE signal peptide analogs as N-formyl PhoE signal peptide, PhoE signal peptide +(1-7) and PhoE signal peptide Val-8----Trp-8 show the same lipid preference as PhoE signal peptide . On the other hand the affinity for an anionic lipid interface is strongly reduced for PhoE signal peptide Lys-19,-20----Asp-19,-20, which correlates with the less efficient translocation of PhoE protein carrying this signal sequence . At limiting anionic lipid concentrations there is a temperature and salt effect on the observed interaction, which is related to a conformational change of the peptide . Signal sequences show clearly conformational flexibility in responds to environmental conditions . Under the conditions used in this study FTIR spectra of PhoE signal peptide-DOPG monolayers show a high content of beta-structure and beta-turn. Cell, 1990 Aug 24, 62(4), 733 - 43 Stringent spacing requirements for transcription activation by CRP; Gaston K et al.; The cyclic AMP receptor protein-cAMP complex (CRP-cAMP) binds at a variety of distances upstream of several E . coli promoters and activates transcription . We have constructed a model system in which a consensus CRP binding site is placed at different distances upstream of the melR promoter . CRP-cAMP activates transcription from melR when bound at a number of positions, all of which lie on the same face of the DNA helix . The two distances at which transcription is strongly activated correspond exactly to those at which CRP-cAMP binds upstream of the well-studied galP1 and lac promoters . Footprinting of the synthetic promoters reveals that RNA polymerase makes identical contacts with their -10 regions even though CRP-cAMP binds at a different distance in each case . Kinetic analysis in vitro indicates that CRP-cAMP activates transcription from these promoters in similar but distinct ways . A model is proposed to explain this two-position activation. Cell, 1990 Aug 24, 62(4), 745 - 55 Function of Neurospora mitochondrial tyrosyl-tRNA synthetase in RNA splicing requires an idiosyncratic domain not found in other synthetases; Cherniack AD et al.; Neurospora mitochondrial tyrosyl-tRNA synthetase (mt TyrRS), which is encoded by nuclear gene cyt-18, functions in splicing group I introns . Analysis of intragenic partial revertants of the cyt-18-2 mutant and in vitro mutants of the cyt-18 protein expressed in E . coli showed that splicing activity of the cyt-18 protein is dependent on a small N-terminal domain that has no homolog in bacterial or yeast mt TyrRSs . This N-terminal splicing domain apparently acts together with other regions of the protein to promote splicing . Our findings support the hypothesis that idiosyncratic sequences in aminoacyl-tRNA synthetase may function in processes other than aminoacylation . Furthermore, they suggest that splicing activity of the Neurospora mt TyrRs was acquired after the divergence of Neurospora and yeast, and they demonstrate one mechanism whereby splicing factors may evolve from cellular RNA binding proteins. Biochemistry, 1990 Aug 21, 29(33), 7715 - 27 Absolute action spectrum of E-FADH2 and E-FADH2-MTHF forms of Escherichia coli DNA photolyase; Payne G et al.; Escherichia coli DNA photolyase mediates photorepair of pyrimidine dimers occurring in UV-damaged DNA . The enzyme contains two chromophores, 1,5-dihydroflavin adenine dinucleotide (FADH2) and 5,10-methenyltetrahydrofolylpolyglutamate (MTHF) . To define the roles of the two chromophores in the photochemical reaction(s) resulting in DNA repair and the effect of DNA structure on the photocatalytic step, we determined the absolute action spectra of the enzyme containing only FADH2 (E-FADH2) or both chromophores (E-FADH2-MTHF), with double- and single-stranded substrates and with substrates of different sequences in the immediate vicinity of the thymine dimer . We found that the shape of the action spectrum of E-FADH2 matches that of the absorption spectrum with a quantum yield phi (FADH2) = 0.69 . The action spectrum of E-FADH2-MTHF is also in a fairly good agreement with the absorption spectrum with phi (FADH2-MTHF) = 0.59 . From these values and from the previously established properties of the two chromophores, we propose that MTHF transfers energy to FADH2 with a quantum yield of phi epsilon T = 0.8 and that 1FADH2 singlet transfers an electron to or from the dimer with a quantum yield phi ET = 0.69 . The chemical nature of the chromophores did not change after several catalytic cycles . The enzyme repaired a thymine dimer in five different sequence contexts with the same efficiency . Similarly, single- and double-stranded DNAs were repaired with the same overall quantum yield. Biochemistry, 1990 Aug 21, 29(33), 7691 - 701 Structural consequences of effector binding to the T state of aspartate carbamoyltransferase: crystal structures of the unligated and ATP- and CTP-complexed enzymes at 2.6-A resolution; Stevens RC et al.; The crystal structure of Escherichia coli aspartate carbamoyltransferase complexed with adenosine 5'-triphosphate (ATP) has been solved by molecular replacement and has been refined to a crystallographic residual of 0.17 at 2.6-A resolution by using the computer program X-PLOR . The unit cell dimensions of this crystal form are a = b = 122.2 A and c = 143.3 A and the space group is P321 . Although the c-axis unit cell dimension is approximately 1 A longer than the corresponding dimension of the CTP-ligated P321 crystal form (c = 142.2 A), the ATP-ligated enzyme adopts a T-like quaternary structure . The base moiety of ATP interacts with residues Glu10, Ile12, and Lys60 while the ribose is near Asp19 and Lys60; the triphosphate entity is bound to Lys94, although His20 and Arg96 are nearby . We observe a higher occupancy for ATP in the allosteric site of the R1 regulatory chain in comparison to the occupancy of the R6 allosteric site . These crystallographically independent sites are related by a molecular 2-fold axis . There are other violations of the noncrystallographic symmetry that are similar to those observed in the refined CTP-ligated aspartate carbamoyltransferase structure . These infringements on the molecular symmetry might be the result of intermolecular interactions in the crystal . To ensure the most meaningful comparison with the ATP-ligated structure, we refined the previously reported CTP-bound and unligated structures to crystallographic residuals between 0.17 and 0.18 using X-PLOR . These X-PLOR refined structures are not significantly different from the initial structures that had been crystallographically refined by a restrained least-squares method . After making all possible comparisons between the CTP- and ATP-ligated and the unligated T-state structures, we find that the most significant differences are located at the allosteric sites and in small changes in the quaternary structures . At the allosteric site, the binding of CTP and ATP successively enlarges the nucleotide binding cavity, particularly in the vicinity of the base . The changes in the quaternary structure can be characterized by an increase in the separation of the catalytic trimers by approximately 0.5 A as ATP binds to the unligated T structure . On the basis of these structural studies, we discuss the relationships between the conformational differences in the allosteric site and the small changes in the quaternary structure within the T form to the possible mechanisms for CTP inhibition and ATP activation. Biochemistry, 1990 Aug 21, 29(33), 7666 - 76 Inhibition of recA protein promoted ATP hydrolysis . 1 . ATP gamma S and ADP are antagonistic inhibitors; Lee JW et al.; ADP and adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) inhibit recA protein promoted ATP hydrolysis by fundamentally different mechanisms . In both cases, at least two modes of inhibition are observed . For ADP, the first mode is competitive inhibition . The second mode is manifested by dissociation of recA protein from DNA . These are readily distinguished in a comparison of ATP hydrolyses that are activated by (a) DNA and (b) high (approximately 2 M) salt concentrations . Competitive inhibition with a significant degree of cooperativity is observed under both sets of conditions, although the DNA-dependent activity is more sensitive to ADP than the high-salt reaction . The reaction in the presence of poly(deoxythymidylic acid) or duplex DNA ceases when about 60% of the available ATP is hydrolyzed, reflecting an ADP-mediated dissociation of recA protein from the DNA that is governed by the ADP/ATP ratio . In contrast, ATP hydrolysis proceeds nearly to completion at high salt concentrations . At high concentrations of ATP and ATP gamma S, ATP gamma S also acts as a competitive inhibitor . At low concentrations of ATP gamma S and ATP, however, ATP gamma S activates ATP hydrolysis . These patterns are observed for recA-mediated ATP hydrolysis with either high salt concentrations or a poly(deoxythymidylic acid) {poly(dT)} cofactor, although the activation is observed at much lower ATP and ATP gamma S concentrations when poly(dT) is used . ATP gamma S can also relieve the inhibitory effect of ADP under some conditions . ATP gamma S and ADP are antagonistic inhibitors, reinforcing the idea that they stabilize different conformations of the protein and suggesting that these conformations are mutually exclusive . The ATP gamma S (ATP) conformation is active in ATP hydrolysis . The ADP conformation is inactive. Biochemistry, 1990 Aug 21, 29(33), 7563 - 71 An essential proline in lambda repressor is required for resistance to intracellular proteolysis; Reidhaar-Olson JF et al.; Pro78 is a solvent-exposed residue at the N-terminal end of alpha-helix 5 in the DNA binding domain of lambda repressor . Random mutagenesis experiments have suggested that Pro78 is essential {Reidhaar-Olson, J.F., & Sauer, R.T . (1990) Proteins: Struct., Funct., Genet . (in press)} . To investigate the requirement for proline at this position, we constructed and studied the properties of a set of ten position 78 mutant proteins . All of these mutants have decreased intracellular activities and are expressed at significantly lower levels than wild type . Pulse-chase experiments show that the mutant proteins are rapidly degraded in the cell; the mutants examined had half-lives of 11-35 min, whereas the wild-type protein has a half-life of greater than 10 h . The rapid degradation of position 78 mutants is not suppressed by mutations that affect known Escherichia coli proteases . The Pro78----Ala mutant could be overexpressed in a dnaJ- strain and was purified . This mutant has full DNA binding activity in vitro, suggesting that its folded structure and ability to form active dimers are similar to those of wild type . The PA78 mutant (Tm = 48 degrees C) is less thermally stable than wild type (Tm = 55 degrees C) . Double-mutant studies show that this instability contributes to but is not the main cause of its rapid intracellular degradation and also suggest that proteolysis proceeds from the denatured forms of proteins containing the PA78 substitution . The PA78 mutation does not appear to introduce a new cleavage site for cellular proteases, nor does the mutation enhance susceptibility to proteases such as thermolysin and trypsin in vitro.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1990 Aug 21, 29(33), 7557 - 63 Mutation of essential catalytic residues in pig citrate synthase; Alter GM et al.; Two amino acid residues, His274 and Asp375, were replaced singly in the active site of pig citrate synthase (PCS) with Gly274, Arg274, Gly375, Asn375, Glu375, and Gln375 . The nonmutant protein and the mutant proteins were expressed in and purified from Escherichia coli, and the effects of these amino acid substitutions on the overall reaction rate and conformation of the PCS protein were studied by initial velocity and full time course kinetic analysis, behavior during affinity column chromatography, and monoclonal antibody reactivity . Native and mutant proteins purified similarly had a subunit molecular weight of 50,000 and were homologous when examined with 10 independent a-PCS monoclonal IgGs or with a polyclonal anti-PHCS serum . No activity was detected for Asn375 or Gln375 . The kcats of the other purified mutant proteins, however, were decreased by about 10(3) compared to the nonmutant enzyme activity . The Km for oxalacetate was decreased 10-fold in the Glu375 protein and was reduced by half in Gly274 and Arg274 PCSs, while the Km for acetyl-CoA was decreased 2-3-fold in Gly274, Arg274, and Gln375 PCSs . A mechanism is proposed that electrostatically links His274 and Asp375. FEBS Lett, 1990 Aug 20, 269(1), 26 - 8 The influence of oligonucleotide-effector on the selectivity of sequence specific modification of 16 S rRNA; Zenkova MA et al.; The influence of duplex stabilizing oligonucleotide-effector (oligonucleotide, carrying N-(2-hydroxyethyl)phenazinium residues on both ends), on selectivity of site-directed modification of E . coli 16 S rRNA (1542 nucleotides in length) under the conditions of its secondary structure stability was studied . The constant of cooperative binding of the reagent and the oligonucleotide-effector with 16 S rRNA was determined . The accuracy of modification was shown to double in the presence of 50 microM effector at 5 microM concentration of the reagent. J Mol Biol, 1990 Aug 20, 214(4), 937 - 48 Stereochemical mechanism of action for thymidylate synthase based on the X-ray structure of the covalent inhibitory ternary complex with 5-fluoro-2'-deoxyuridylate and 5,10-methylenetetrahydrofolate; Matthews DA et al.; The structure of the Escherichia coli thymidylate synthase (TS) covalent inhibitory ternary complex consisting of enzyme, 5-fluoro-2'-deoxyuridylate (FdUMP) and 5,10-methylene tetrahydrofolate (CH2-H4PteGlu) has been determined at 2.5 A resolution using difference Fourier methods . This complex is believed to be a stable structural analog of a true catalytic intermediate . Knowledge of its three-dimensional structure and that for the apo enzyme, also reported here, suggests for the first time how TS may activate dUMP and CH2-H4PteGlu leading to formation of the intermediate and offers additional support for the hypothesis that the substrate and cofactor are linked by a methylene bridge between C-5 of the substrate nucleotide and N-5 of the cofactor . By correlating these structural results with the known stereospecificity of the TS-catalyzed reaction it can be inferred that the catalytic intermediate, once formed, must undergo a conformational isomerization before eliminating across the bond linking C-5 of dUMP to C-11 of the cofactor . The elimination itself may be catalyzed by proton transfer to the cofactor's 5 nitrogen from invariant Asp169 buried deep in the TS active site . The juxtaposition of Asp169 and bound tetrahydrofolate in TS is remarkably reminiscent of binding geometry found in dihydrofolate reductase where a similarly conserved carboxyl group serves as a general acid for protonating the corresponding pyrazine ring nitrogen of dihydrofolate. J Mol Biol, 1990 Aug 20, 214(4), 923 - 36 Crystal structure of Escherichia coli thymidylate synthase containing bound 5-fluoro-2'-deoxyuridylate and 10-propargyl-5,8-dideazafolate; Matthews DA et al.; The crystal structure of an Escherichia coli thymidylate synthase (TS) ternary complex containing 5-fluoro-2'-deoxyuridylate (FdUMP) and 10-propargyl-5,8-dideazafolate (PDDF) has been determined and refined at 2.3 A resolution . Each of the two chemically identical subunits folds into a three-layer domain anchored by a large six-stranded mixed beta-sheet . The backside of one sheet is juxtaposed against the corresponding face of the equivalent sheet in the second protomer creating a beta-sandwich . In contrast to other proteins of known structure in which aligned beta-sheets stack face to face with a counterclockwise rotation, sheets in the TS dimer are related by a clockwise twist . The substrate-binding pocket is a large funnel-shaped cleft extending some 25 A into the interior of each subunit and is surrounded by 30 amino acids, 28 from one subunit and two from the other . FdUMP binds at the bottom of this pocket covalently linked through C-6 to the sulfur of Cys146 . Up-pointing faces of the pyrimidine and ribose rings are exposed to provide a complementary docking surface for the quinazoline ring of PDDF . The quinazoline inhibitor binds in a partially folded conformation with its p-aminobenzoyl glutamate tail exposed at the entrance to the active site cleft . Ternary complex formation is associated with a large conformational change involving four residues at the protein's carboxy terminus that close down on the distal side of the inhibitor's quinazoline ring, capping the active site and sequestering the bound ligands from bulk solvent. J Mol Biol, 1990 Aug 20, 214(4), 845 - 64 Closely spaced and divergent promoters for an aminoacyl-tRNA synthetase gene and a tRNA operon in Escherichia coli . Transcriptional and post-transcriptional regulation of gltX, valU and alaW; Brun YV et al.; The transcription of the gltX gene encoding the glutamyl-tRNA synthetase and of the adjacent valU and alaW tRNA operons of Escherichia coli K-12 has been studied . The alaW operon containing two tRNA(GGCAla) genes, is 800 base-pairs downstream from the gltX terminator and is transcribed from the same strand . The valU operon, containing three tRNA(UACVal) and one tRNA(UUULys) (the wild-type allele of supN) genes, is adjacent to gltX and is transcribed from the opposite strand . Its only promoter is upstream from the gltX promoters . The gltX gene transcript is monocistronic and its transcription initiates at three promoters, P1, P2 and P3 . The transcripts from one or more of these promoters are processed by RNase E to generate two major species of gltX mRNA, which are stable and whose relative abundance varies with growth conditions . The stability of gltX mRNA decreases in an RNase E- strain and its level increases with growth rate about three times more than that of the glutamyl-tRNA synthetase . The 5' region of these mRNAs can adopt a stable secondary structure (close to the ribosome binding site) that is similar to the anticodon and part of the dihydroU stems and loops of tRNA(Glu), and which might be involved in translational regulation of GluRS synthesis . The gltX and valU promoters share the same AT-rich and bent upstream region, whose position coincides with the position of the upstream activating sequences of tRNA and rRNA promoters to which they are similar . This suggests that gltX and valU share transcriptional regulatory mechanisms. J Mol Biol, 1990 Aug 20, 214(4), 825 - 43 Precise mapping and comparison of two evolutionarily related regions of the Escherichia coli K-12 chromosome . Evolution of valU and lysT from an ancestral tRNA operon; Brun YV et al.; Two tRNA operons have been found near the gltX gene encoding the glutamyl-tRNA synthetase of Escherichia coli K-12 . The alaW operon previously undetected from genetic data and containing two identical tRNA(GGCAla) genes is 800 base-pairs downstream from the gltX terminator and is transcribed from the same strand . The valU operon containing genes for three identical tRNA(UACVal) and one tRNA(UUULys) (the wild-type allele of supN), is adjacent to gltX and is transcribed from the opposite strand . Five open reading frames were also found in this region encoding putative polypeptides of 62, 105, 130, 167 and 294 amino acid residues . ORF294 is a new member of the lysR family of bacterial transcriptional activators . The possibility that this is the xapR gene is discussed . Comparison of the physical and linkage maps of the E . coli chromosome in the 52 minute region has permitted precise mapping of most of the 18 genes in this region with the order nupC-glk- less than (alaW beta-ala W alpha)-1 kb- less than gltX-0.3 kb-(valU alpha-valU beta-valU gamma-lysV = supN) greater than xapR-xapA- less than lig-1 kb-cysK greater than -0.4 kb-ptsH greater than -0.05 kb-pstI greater than -0.05 kb-crr greater than -cysM-cysA in the clockwise order (greater than and less than indicate the direction of transcription; kb, 10(3) bases) . The last two genes of valU (52 min) and lysT (16.5 min) are arranged in a similar fashion and a highly conserved region has been found in both operons . This suggests that the valU and lysT operons probably arose by a duplication of an ancestral tRNA operon . This is the first example of what may be two different tRNA operons from the same organism evolving from an ancestral tRNA gene . Comparison of the 16 and 52 minute regions of the E . coli K-12 chromosome suggests that these two regions could share a common ancestor. FEBS Lett, 1990 Aug 20, 269(1), 96 - 100 SecE-dependent overproduction of SecY in Escherichia coli . Evidence for interaction between two components of the secretory machinery; Matsuyama S et al.; The secY and secE genes were individually cloned and placed under the control of the tac promoter on plasmids . Induction with isopropyl-beta-D-thiogalactopyranoside resulted in the overproduction of SecE, but not that of SecY . The simultaneous induced expression of both genes in the same cells resulted in the overproduction of SecY together with that of SecE . SecY and SecE thus overproduced were localized in the cytoplasmic membrane as those expressed at the normal levels were . It is suggested that SecY and SecE interact with each other in the cytoplasmic membrane . The numbers of the SecY and SecE molecules per cell were estimated. FEBS Lett, 1990 Aug 20, 269(1), 113 - 6 A protein with sequence identity to Skp (FirA) supports protein translocation into plasma membrane vesicles of Escherichia coli; Thome BM et al.; We have purified to homogeneity a 15 kDa-protein from a ribosomal salt extract of Escherichia coli that compensates in vitro a defect of SecA but not of SecB . Removal of this protein from a cell-free transcription/translation system impairs translocation into plasma membrane vesicles of the precursors of LamB and to a lesser degree also of OmpA . These results suggest a role of the 15 kDa-protein in bacterial protein export . The NH2-terminal 35 amino acids were found to be identical to those of the skp (firA) gene product, to which several putative functions have previously been attributed. J Mol Biol, 1990 Aug 20, 214(4), 885 - 96 Tropomyosin has discrete actin-binding sites with sevenfold and fourteenfold periodicities; Hitchcock-DeGregori SE et al.; Analysis of the periodic distribution of amino acids in tropomyosin has revealed the presence of seven or 14 quasi-equivalent actin-binding sites . We tested the hypothesis of periodic actin-binding sites by making deletions of chicken striated alpha-tropomyosin cDNA using oligonucleotide-directed mutagenesis . The deletions corresponded to one-half (amino acid residues 47 to 67), two-thirds (residues 47 to 74) and one actin-binding site (residues 47 to 88), on the basis of there being seven sites . The mutant cDNAs were expressed as fusion and non-fusion proteins in Escherichia coli and analyzed for actin binding and regulatory function . Fusion tropomyosin binds to actin with an affinity similar to that of muscle tropomyosin . Of the mutant fusion tropomyosins, only that with a full site deleted retained actin affinity and the ability to inhibit the actomyosin S1 ATPase, though it was less effective than wild-type . We conclude that an integral number of half-turns of the tropomyosin coiled-coil, and the consequential sevenfold periodicity, as well as the correct orientation of the ends with respect to each other, are important for actin binding . On the other hand, non-fusion tropomyosin binds well to actin only in the presence of troponin, and the binding is calcium-sensitive . Assay of non-fusion mutant tropomyosins showed that mutants with deletion of one-half and one actin binding site both had high affinity for actin, equal to or slightly less than wild-type . The ability of these two mutants to regulate the actomyosin or acto-S1 ATPase with troponin in the absence of calcium was indistinguishable from that of the wild-type . The normal regulatory function of the mutant with a 1/14 deletion (removal of a quarter turn or half a site) indicates that a 14-fold periodicity is adequate for regulation, consistent with the presence of two sets of seven alpha and seven beta quasi-equivalent actin-binding sites . An alternative explanation is that the alpha-sites are of primary importance and that proper alignment of the alpha-sites in every second tropomyosin, as when half a site is deleted, is sufficient for normal regulatory function . Deletion of a non-integral period (2/3 of a site) severely compromised actin-binding and regulatory function, presumably due to the inability of the mutant to align properly on the actin filament. FEBS Lett, 1990 Aug 20, 269(1), 60 - 4 Characterization of a chemically synthesized RNA having the sequence of the yeast initiator tRNA(Met); Bratty J et al.; A 75-unit long oligoribonucleotide corresponding to the sequence of the Saccharomyces cerevisiae initiator tRNA was synthesized chemically . The crude RNA was purified, and the sequence was verified by RNA sequencing techniques . A particularly useful purification step involved hydrophobic chromatography on BND-cellulose . The purified RNA could be aminoacylated to 28% of a bona fide initiator tRNA(Met) sample and threonylated to 76% of the level observed with native tRNA(fMet) from E . coli. Eur J Biochem, 1990 Aug 17, 191(3), 701 - 4 Fructose-1,6-bisphosphate-activated pyruvate kinase from Escherichia coli . Nature of bonds involved in the allosteric mechanism; Speranza ML et al.; The allosteric properties of the fructose-1,6-bis-phosphate-activated pyruvate kinase from Escherichia coli were examined in the presence of a number of fructose bisphosphate analogues, as well as of increased ionic strength (NaCl) and of the hydrogen-bond-breaking agent, formamide . Fructose 2,6-bisphosphate, ribulose 1,5-bisphosphate and 5-phosphorylribose 1-pyrophosphate gave allosteric activation (additive to that of fructose 1,6-bisphosphate) . Formamide always decreased Vmax, but left unchanged the Km for phosphoenolpyruvate, while it decreased the concentration of fructose bisphosphate required to give half-maximal activity (K0.5) . NaCl increased the K0.5 for both phosphoenolpyruvate and fructose bisphosphate, leaving Vmax unchanged . These results are consistent with ionic binding of fructose bisphosphate through phosphates and with a critical role of hydrogen bonds in stabilizing both the inactive and the active enzyme conformers. Eur J Biochem, 1990 Aug 17, 191(3), 743 - 53 Escherichia coli DNA polymerase I: inherent exonuclease activities differentiate between monofunctional and bifunctional adducts of DNA and cis- or trans-diamminedichloroplatinum(II) . An exonuclease investigation of the kinetics of the adduct formation; Bernges F et al.; {3H}dGMP-3'-labelled, activated salmon testis DNA and {32P}dGMP-5'-labelled open circular M13 DNA were reacted with cis-diamminedichloroplatinum(II), cis-diamminechloroaquaplatinum(II), cis-diamminediaquaplatinum(II) or trans-diamminechloroaquaplatinum(II) . The reaction was arrested after arbitrary times by adjustment to slightly alkaline solution conditions . The platinum-containing DNA was digested with Escherichia coli DNA polymerase I . The progress of nucleotide release was measured by acid precipitation of undigested DNA . Solubilized nucleotides and adducts were analyzed by HPLC . The 3'-5'-exonuclease activity liberated single-coordinated dGMP-platinum(II) adducts from both cis- and trans-platinum(II) treated salmon testis DNA and a small fraction of adducts of cis-platinum(II) that coordinated two molecules of dGMP . The bisadduct was derived from non-neighboring guanine residues probably located at or close to 3'-termini . This nuclease activity neither cut between nor after neighboring guanine residues crosslinked by cis-platinum(II) . No bisadduct was liberated for trans-platinum(II) . The 5'-3'-exonuclease activity did not liberate any nucleotide adducts from cis-platinum(II)-treated DNa . However, it removed single-coordinated guanine adducts of trans-diamminedichloroplatinum(II) . From the kinetics of the appearance of dGMP monoadducts and the inhibition of digestion, a reaction scheme is formulated for the reaction of platinum(II) complexes with DNA that confirms and extends the previously published one {W . Schaller, H . Reisner & E . Holler (1987) Biochemistry 26, 943-950} . The longevity of the dGMP monoadduct intermediate is discussed in the context of the efficiency of cis-diamminedichloroplatinum(II) as an antitumor drug. Biochim Biophys Acta, 1990 Aug 17, 1035(2), 230 - 6 Characterization of FMN-dependent NADH-quinone reductase induced by menadione in Escherichia coli; Hayashi M et al.; It was found that when Escherichia coli is grown in the presence of 0.2-0.3 mM menadione (2-methyl-1,4-naphthoquinone), an FMN-dependent NADH-quinone reductase increases more than 20-fold in the cytoplasmic fraction . The menadione-induced quinone reductase was isolated from the cytoplasmic fraction of induced cells . The purified enzyme had an Mr of 24 kDa on SDS-polyacrylamide gel electrophoresis . The enzyme required flavin as a cofactor and a half-maximum activity was obtained with 0.54 microM FMN or 16.5 microM FAD . The enzyme had a broad pH optimum at pH 7.0-8.0 and reacted with NADH, but not with NADPH . The reaction followed a ping-pong mechanism and the intrinsic Km values for NADH and menadione were estimated to be 132 microM and 2.0 microM, respectively . Dicoumarol was a simple competitive inhibitor with respect to NADH with a Ki value of 0.22 microM . The electron acceptor specificity of this enzyme was very similar to that of NAD(P)H: (quinone acceptor) oxidoreductase (EC 1.6.99.2, DT-diaphorase) from rat liver . Since menadione is reduced by the two-electron reduction pathway to menadiol, the induction of this enzyme is likely to be an adaptive response of E . coli to partially alleviate the toxicity of menadione. Science, 1990 Aug 17, 249(4970), 783 - 6 External guide sequences for an RNA enzyme; Forster AC et al.; Ribonuclease P (RNase P) from Escherichia coli or its catalytic RNA subunit can efficiently cleave small RNA substrates that lack the conserved features of natural substrates of RNase P if an additional small RNA is also present . This additional RNA must contain a sequence complementary to the substrate {external guide sequence (EGS)} and a 3'-proximal CCA sequence to ensure cleavage . The aminoacyl acceptor stem and some additional 5'- and 3'-terminal sequences of a precursor transfer RNA are sufficient to allow efficient cleavage by RNAase P, and the 2'-hydroxyl group at the cleavage site is not absolutely necessary for cleavage . In principle, any RNA could be targeted by a custom-designed EGS RNA for specific cleavage by RNase P in vitro or in vivo. Biochem Biophys Res Commun, 1990 Aug 16, 170(3), 1319 - 24 Thyroid hormone binding protein contains glycosylation site binding protein activity; Kimura H et al.; Several lines of evidence provided by other workers indicate that within the same species thyroid hormone binding protein, the beta-subunit of prolyl hydroxylase, and protein disulfide isomerase are the same protein . We sought to determine if glycosylation site binding protein, a lumenal protein of the endoplasmic reticulum, also has the same primary structure . To accomplish this the level of glycosylation site binding protein (GSBP) activity, measured by photolabeling with a glycosylation site peptide probe, was carried out in preparations of 3T3 cells and in E . coli transformed with human thyroid hormone binding protein cDNA . The results strongly support the idea that GSBP is identical to these other lumenal proteins of the endoplasmic reticulum. Biochem Biophys Res Commun, 1990 Aug 16, 170(3), 1294 - 300 Site-specific mutagenesis of PRD1 DNA polymerase: mutations in highly conserved regions of the family B DNA polymerase; Jung GH et al.; The PRD1 DNA polymerase is a small multifunctional enzyme containing three major conserved amino acid sequences shared by family B DNA polymerases . Thus, the PRD1 DNA polymerase provides an useful model system with which to study structure-function relationships of DNA polymerase molecules . In order to investigate the functional and structural roles of the highly conserved amino acid sequences, we have introduced mutations into each of the 3 conserved regions of the PRD1 DNA polymerase . Genetic complementation study as well as DNA polymerase assay indicated that each mutation inactivated DNA polymerase catalytic activity, but not the 3' to 5' exonuclease activity. J Biol Chem, 1990 Aug 15, 265(23), 13618 - 22 Identification and initial characterization of translational initiation factor 2 from bovine mitochondria; Liao HX et al.; The bovine liver mitochondrial factor that promotes the binding of fMet-tRNA to mitochondrial ribosomes, initiation factor 2 (IF-2mt), has been identified in the postribosomal supernatant fraction of isolated liver mitochondria . This factor has been purified approximately 5,000-fold and present preparations are estimated to be about 10% pure . IF-2mt has an apparent molecular weight of about 140,000 as determined by gel filtration chromatography . IF-2mt is active in stimulating fMet-tRNA binding to Escherichia coli ribosomes but E . coli IF-2 is not active in promoting initiator tRNA binding to animal mitochondrial ribosomes . The IF-2mt-mediated binding of fMet-tRNAi(Met) to mitochondrial ribosomes is dependent on the presence of a message such as poly(A,U,G) and on GTP . Nonhydrolyzable analogs of GTP are 2-3-fold less effective in promoting initiation complex formation on mitochondrial ribosomes than is GTP suggesting that IF-2mt is capable of recycling to some extent under the current assay conditions. Anal Biochem, 1990 Aug 15, 189(1), 138 - 41 An ultrafiltration assay for nucleotide binding to ribonucleotide reductase; Ormo M et al.; Direct partition through ultrafiltration was applied to develop a method for the study of nucleotide binding to ribonucleotide reductase from Escherichia coli . The assay involved a 0.5- to 1-min centrifugation step where bound and unbound nucleotides are separated over an ultrafiltration membrane . No effects were seen due to hyperconcentration of protein at the membrane surface . The method was verified by measuring binding of dATP, ATP, dTTP, dGTP, and GDP at 25 and 4 degrees C with dissociation constants ranging from 0.1 to 80 microM . The results were in good agreement with earlier data obtained by other techniques and extend our knowledge in the case of ATP and dGTP binding at 25 degrees C. Biochem J, 1990 Aug 15, 270(1), 141 - 8 The organization of open complexes between Escherichia coli RNA polymerase and DNA fragments carrying promoters either with or without consensus -35 region sequences; Chan B et al.; Transcription initiation at the Escherichia coli galP1 promoter does not depend on specific nucleotide sequences in the -35 region . Footprint analysis of transcriptionally competent complexes between E . coli RNA polymerase and DNA fragments carrying galP1 shows that RNA polymerase protects sequences as far upstream as -55, whereas sequences around the -35 region are exposed . In contrast, with galP1 derivatives carrying -35 region sequences resembling the consensus, RNA polymerase protects bases as far as -45, and the -35 region is fully protected . Taken together, our data suggest that the overall architecture of RNA polymerase-promoter complexes can vary according to whether or not consensus -35 region sequences are present; in the absence of these sequences, open complex formation requires distortion of the promoter DNA . However, the unwinding of promoter DNA around the transcription start is not affected by the nature of the -35 region sequence . With a galP1 derivative carrying point mutations in the spacer region that greatly reduce promoter activity, the protection of bases by RNA polymerase around the -10 sequence and transcription start site is reduced . In contrast, protection of the region upstream of -25 is unaffected by the spacer mutations, although sequences from -46 to -54 become hypersensitive to attack by potassium permanganate, indicating severe distortion or kinking of this zone . We suggest that, with this galP1 derivative, RNA polymerase is blocked in a complex that is an intermediate on the path to open complex formation. J Biol Chem, 1990 Aug 15, 265(23), 14023 - 9 Physicochemical properties of the lipopolysaccharide unit that activates B lymphocytes; Takayama K et al.; We have examined the physical state of highly purified deep rough chemotype lipopolysaccharide (ReLPS) from Escherichia coli D31m4 as an aqueous suspension and as complexes with bovine serum albumin min (BSA) . The ReLPS suspension showed large ellipsoidal particles 12-38 nm wide and 40-100 nm long . The solubility of this form of ReLPS was determined by equilibrium dialysis experiments to be 3.3 x 10(-8) M at 22 degrees C and 2.8 x 10(-8) M at 37 degrees C in 150 mM Tris-KCl, pH 7.5; 3.0 x 10(-8) M at 37 degrees C in 0.75 mM Tris-KCl, pH 7.5 . The BSA-ReLPS complexes were fractionated on a Sephacryl S-200 column to yield peaks I and II with apparent masses of about 240 and 70 kDa, respectively . Peak II was a BSA monomer with estimated BSA:ReLPS molar ratios of 1:1-1:7 . The ReLPS suspension and the two complexes were compared as antigens in enzyme-linked immunosorbent assays using three select monoclonal antibodies to lipopolysaccharide . The results were consistent with the high state of disaggregation of the ReLPS in both peaks I and II . Since the ReLPS in these complexes were not visible by electron microscopy, they did not contain vesicles or large particles . All forms of ReLPS tested were capable of stimulating 70Z/3, a lipopolysaccharide-responsive murine pre-B cell line . However, peak II was consistently more stimulatory at very low concentrations than the other preparations . The maximally stimulatory concentration of ReLPS for 70Z/3 cells was 40 ng/ml (1.6 x 10(-8) M) for peak II and 70 ng/ml (2.8 x 10(-8) M) for the ReLPS suspension . As expected, the above concentrations were at or below the solubility of the ReLPS . These results suggested that the highly disaggregated form of ReLPS (possibly the monomer) is the active unit that stimulates the cellular response in 70Z/3 cells. J Biol Chem, 1990 Aug 15, 265(23), 14016 - 22 Three-dimensional structure of thymidine phosphorylase from Escherichia coli at 2.8 A resolution; Walter MR et al.; The three-dimensional structure of thymidine phosphorylase from Escherichia coli has been determined at 2.8 A resolution using multiple-isomorphous-replacement techniques . The amino acid sequence deduced from the deoA DNA sequence is also reported . Thymidine phosphorylase exists in the crystal as an S-shaped dimer in which the subunits are related by a crystallograp