Biochemistry, 1991 Apr 23, 30(16), 4055 - 61 Specificities and kinetics of uracil excision from uracil-containing DNA oligomers by Escherichia coli uracil DNA glycosylase; Varshney U et al.; Uracil DNA glycosylase excises uracil residues from DNA that can arise as a result of deamination of cytosine or incorporation of dUMP residues by DNA polymerase . We have carried out a detailed study to define the specificities and the kinetic parameters for its substrates by using a number of synthetic oligodeoxyribonucleotides of varying lengths and containing uracil residue(s) in various locations . The results show that the Escherichia coli enzyme can remove a 5'-terminal U from an oligomer only if the 5'-end is phosphorylated . The enzyme does not remove U residues from a 3'-terminal position, but U residues can be excised from oligonucleotides with either pd(UN)p or pd(UNN) 3'-termini . The oligomer d(UUUUT) can have the second or third U residues from the 5'-end excised even when the neighboring site is an abasic site (3' or 5', respectively) . On the basis of these findings, pd(UN)p was anticipated to be the smallest size substrate . Results show detectable amounts of U release from the substrate pd(UT)p; however, significantly higher amounts of U release were observed from pd(UT-sugar) or pd(UTT) . Determinations of the Km and Vmax values show that the different rates of U excision from oligomers of different sizes (trimeric to pentameric) but containing U in the same position are largely due to the differences in the Km values, whereas the different rates of U excision from the substrates of the same size but containing U in different positions are largely due to different Vmax values. Biochemistry, 1991 Apr 23, 30(16), 4009 - 20 Two new photoaffinity polyamines appear to alter the helical twist of DNA in nucleosome core particles; Clark E et al.; Two new photoaffinity derivatives of polyamines have been synthesized by the reaction of spermine or spermidine with methyl 4-azidobenzimidate . The new compounds were purified chromatographically and characterized by several methods including proton magnetic resonance spectroscopy . The spermine derivative is N1-ABA-spermine {(azidobenzamidino)spermine}, and the spermidine derivative is a mixture of N1- and N8-ABA-spermidine . ABA-spermine stabilizes nucleosome core particles in thermal denaturation experiments, with similar but not identical effects when compared with the parent polyamine, spermine . In circular dichroism experiments, ABA-spermine was capable of producing a B----Z transition in poly(dG-m5dC) at a concentration of 30 microM, compared with 5 microM required to produce the same effect with spermine . On the other hand, ANB-spermine {(azidonitrobenzoyl)spermine; Morgan, J . E., Calkins, C . C., & Matthews, H . R . (1989) Biochemistry 28, 5095-5106} stabilized the B form of poly(dG-br5dC) . ABA-spermine is a potent inhibitor of ornithine decarboxylase from Escherichia coli, giving 50% inhibition at 0.12 mM, while ANB-spermine is a modest inhibitor, comparable to spermine or spermidine . Under conditions of nitrogen-limited growth, yeast take up ABA-spermine and ABA-spermidine at approximately one-third to half the rate of spermidine or spermine . In contrast, ANB-spermine was not significantly taken up . The photoaffinity polyamines were used to photoaffinity label the DNA in nucleosome core particles, and the sites of labeling were determined by exonuclease protection . All photoaffinity reagents showed both nonspecific labeling and specific sites of higher occupancy . However, the positions of the sites varied: the ANB-spermine sites confirmed those previously reported (Morgan et al., 1989); the ABA-spermine and ABA-spermidine sites were spaced at 9.8 bp intervals from the 3' end of each DNA strand . This observation, together with the effect of spermine on the circular dichroism of DNA in nucleosome core particles, implies that polyamines alter the helical twist of DNA in nucleosome core particles . The ABA-polyamines are offered as general-purpose photoaffinity polyamine reagents. Biochemistry, 1991 Apr 23, 30(16), 4072 - 7 Effect of substitution of a lysyl residue that binds pyridoxal phosphate in thermostable D-amino acid aminotransferase by arginine and alanine; Nishimura K et al.; Lys-145 of the thermostable D-amino acid aminotransferase, which binds pyridoxal phosphate, was replaced by Ala or Arg by site-directed mutagenesis . Both mutant enzymes were purified to homogeneity; their absorption spectra indicated that both mutant enzymes contained pyridoxal phosphate bound non-covalently . Even though the standard assay method did not indicate any activity with either mutant, addition of an amino donor, D-alanine, to the Arg-145 mutant enzyme led to a slow decrease in absorption at 392 nm with a concomitant increase in absorption at 333 nm . This result suggests that the enzyme was converted into the pyridoxamine phosphate form . The amount of pyruvate formed was almost equivalent to that of the reactive pyridoxal phosphate in the mutant enzyme . Thus, the Arg-145 mutant enzyme is able to catalyze slowly the half-reaction of transamination . Exogenous amines, such as methylamine, had no effect on the half-reaction with the Arg-145 mutant enzyme . In contrast, the Ala-145 mutant enzyme neither underwent the spectral change by addition of D-alanine nor catalyzed pyruvate formation, in the absence of added amine . However, the Ala-145 mutant enzyme catalyzed the half-reaction significantly in the presence of added amine . These findings suggest that a basic amino acid residue, such as lysine or arginine, is required at position 145 for catalysis of the half-reaction . The role of the exogenous amines differs with various active-site mutant enzymes. Biochemistry, 1991 Apr 23, 30(16), 3936 - 42 Properties of the two terminal oxidases of Escherichia coli; Puustinen A et al.; Proton translocation coupled to oxidation of ubiquinol by O2 was studied in spheroplasts of two mutant strains of Escherichia coli, one of which expresses cytochrome d, but not cytochrome bo, and the other expressing only the latter . O2 pulse experiments revealed that cytochrome d catalyzes separation of the protons and electrons of ubiquinol oxidation but is not a proton pump . In contrast, cytochrome bo functions as a proton pump in addition to separating the charges of quinol oxidation . E . coli membranes and isolated cytochrome bo lack the CuA center typical of cytochrome c oxidase, and the isolated enzyme contains only 1Cu/2Fe . Optical spectra indicate that high-spin heme o contributes less than 10% to the reduced minus oxidized 560-nm band of the enzyme . Pyridine hemochrome spectra suggest that the hemes of cytochrome bo are not protohemes . Proteoliposomes with cytochrome bo exhibited good respiratory control, but H+/e- during quinol oxidation was only 0.3-0.7 . This was attributed to an "inside out" orientation of a significant fraction of the enzyme . Possible metabolic benefits of expressing both cytochromes bo and d in E . coli are discussed. Biochemistry, 1991 Apr 23, 30(16), 3893 - 8 Interaction of the membrane-bound D-lactate dehydrogenase of Escherichia coli with phospholipid vesicles and reconstitution of activity using a spin-labeled fatty acid as an electron acceptor: a magnetic resonance and biochemical study; Truong HT et al.; The interaction with phospholipid vesicles of the membrane-bound respiratory enzyme D-lactate dehydrogenase of Escherichia coli has been studied . Proteolytic digestion studies show that D-lactate dehydrogenase is protected from trypsin digestion to a larger extent when it interacts with phosphatidylglycerol than with phosphatidylcholine vesicles . Wild-type D-lactate dehydrogenase and mutants in which an additional tryptophan is substituted in selected areas by site-specific oligonucleotide-directed mutagenesis have been labeled with 5-fluorotryptophan . 19F nuclear magnetic resonance studies of the interaction of these labeled enzymes with small unilamellar phospholipid vesicles show that Trp 243, 340, and 361 are exposed to the lipid phase, while Trp 384, 407, and 567 are accessible to the external aqueous phase . Reconstitution of enzymatic activity in phospholipid vesicles has been studied by adding enzyme and substrate to phospholipid vesicles containing a spin-labeled fatty acid as an electron acceptor . The reduction of the doxyl group of the spin-labeled fatty acid has been monitored indirectly by nuclear magnetic resonance and directly by electron paramagnetic resonance . These results indicate that an artificial electron-transfer system can be created by mixing D-lactate dehydrogenase and D-lactate together with phospholipid vesicles containing spin-labeled fatty acids. Biochemistry, 1991 Apr 23, 30(16), 3834 - 40 Isolation and characterization of functional domains of UvrA; Myles GM et al.; The sequence of Escherichia coli UvrA protein suggests that it may fold into two functional domains each possessing DNA binding and ATPase activities . We have taken two approaches to physically isolate polypeptides corresponding to the two putative domains . First, a 180 base pair DNA segment encoding multiple collagenase recognition sequences was inserted into UvrA's putative interdomain hinge region . This UvrA derivative was purified and digested with collagenase, and the resulting 70-kDa N-terminal and 35-kDa C-terminal fragments were purified . Both fragments possessed nonspecific DNA binding activity, but only the N-terminal domain retained its nucleotide binding capacity as evidence by measurements of ATP hydrolysis and by ATP photo-cross-linking . Together, the two fragments failed to substitute for UvrA in reconstituting (A)BC excinuclease and, therefore, were presumed to be unable to load UvrB onto damaged DNA . Second, the DNA segments encoding the two domains were fused to the beta-galactosidase gene . The UvrA N-terminal domain-beta-galactosidase fusion protein was overproduced and purified . This fusion protein had ATPase activity, thus confirming that the amino-terminal domain does possess an intrinsic ATPase activity independent of any interaction with the carboxy terminus . Our results show that UvrA has two functional domains and that the specificity for binding to damaged DNA is provided by the proper three-dimensional orientation of one zinc finger motif relative to the other and is not an intrinsic property of an individual zinc finger domain. Biochemistry, 1991 Apr 23, 30(16), 3824 - 34 Site-specific mutagenesis of conserved residues within Walker A and B sequences of Escherichia coli UvrA protein; Myles GM et al.; UvrA is the ATPase subunit of the DNA repair enzyme (A)BC excinuclease . The amino acid sequence of this protein has revealed, in addition to two zinc fingers, three pairs of nucleotide binding motifs each consisting of a Walker A and B sequence . We have conducted site-specific mutagenesis, ATPase kinetic analyses, and nucleotide binding equilibrium measurements to correlate these sequence motifs with activity . Replacement of the invariant Lys by Ala in the putative A sequences indicated that K37 and K646 but not K353 are involved in ATP hydrolysis . In contrast, substitution of the invariant Asp by Asn in the B sequences at positions D238, D513, or D857 had little effect on the in vivo activity of the protein . Nucleotide binding studies revealed a stoichiometry of 0.5 ADP/UvrA monomer while kinetic measurements on wild-type and mutant proteins showed that the active form of UvrA is a dimer with 2 catalytic sites which interact in a positive cooperative manner in the presence of ADP; mutagenesis of K37 but not of K646 attenuated this cooperativity . Loss of ATPase activity was about 75% in the K37A, 86% in the K646A mutant, and 95% in the K37A-K646A double mutant . These amino acid substitutions had only a marginal effect on the specific binding of UvrA to damaged DNA but drastically reduced its ability to deliver UvrB to the damage site . We find that the deficient UvrB loading activity of these mutant UvrA proteins results from their inability to associate with UvrB in the form of (UvrA)2(UvrB)1 complexes . We conclude that UvrA forms a dimer with two ATPase domains involving K37 and K646 and that the work performed by ATP hydrolysis is the delivery of UvrB to the damage site on DNA. Eur J Biochem, 1991 Apr 23, 197(2), 331 - 6 Pumpkin malate synthase . Cloning and sequencing of the cDNA and northern blot analysis; Mori H et al.; A cDNA clone encoding the glyoxysomal malate synthase (EC 4.1.3.2) was identified by immunoscreening of a cDNA expression library constructed from poly(A)-rich RNA of etiolated pumpkin cotyledons . Determination of the DNA sequence of the 1979-nucleotide cDNA revealed a 1698-nucleotide open reading frame that encodes a polypeptide of 64632 Da . The identification of the cDNA for malate synthase was confirmed by matching three sequences obtained by peptide-sequence analyses of fragments generated by acid treatment of the purified enzyme . Northern blot analysis revealed that the probe hybridized to a single 2.3-kb species of mRNA species from etiolated pumpkin cotyledons which was not present in green pumpkin cotyledons . In a comparison of deduced amino acid sequences, pumpkin malate synthase was found to exhibit 83% and 48% similarity to the malate synthases from rape and Escherichia coli, respectively . Based on the amino acid sequence similarity and the hydropathy profiles of these three malate synthases, the signal for targeting the enzyme to microbodies is discussed. Biochemistry, 1991 Apr 23, 30(16), 3841 - 9 Noncovalent drug-DNA binding interactions that inhibit and stimulate (A)BC excinuclease; Selby CP et al.; (A)BC excinuclease from Escherichia coli catalyzes the initial step of nucleotide excision repair . It recognizes and binds to many types of covalent modifications in DNA and incises the damaged strand on both sides of the lesion . We employed a variety of noncovalent DNA binding drugs to examine in vitro the mechanisms and the nature of the DNA-drug interactions responsible for two phenomena: inhibition of excision repair by caffeine and other noncovalent DNA binding compounds; incision of undamaged DNA produced by (A)BC excinuclease in the presence of the bisintercalating drug ditercalinium . All of the chemicals examined (e.g., actinomycin D, caffeine, ethidium bromide, and Hoechst 33258) inhibited incision of a covalent adduct by (A)BC excinuclease, and direct evidence is given for a common mechanism in which UvrA is depleted by binding to drug-undamaged DNA complexes . In the absence of significant amounts of undamaged DNA, another mechanism of inhibition was observed, in which enzyme bound to noncovalent drug-DNA complexes in the vicinity of the lesion prevents formation of preincision complexes at the lesion . Ditercalinium and unexpectedly all of the other drugs examined promoted the incision of undamaged DNA when the enzyme was present at high concentration . Thus, this activity contrary to previous assumptions is not unique to bisintercalators . Another unexpected finding was stimulation of incision at certain sites of photodamage in DNA produced by low concentrations of noncovalent DNA binding chemicals.(ABSTRACT TRUNCATED AT 250 WORDS) FEBS Lett, 1991 Apr 22, 282(1), 209 - 13 Sequence conservation in the alpha and beta subunits of pyruvate dehydrogenase and its similarity to branched-chain alpha-keto acid dehydrogenase; Wexler ID et al.; Amino acid sequence comparison of 8 alpha and 6 beta subunits of the alpha-keto acid dehydrogenase (E1) component of the pyruvate dehydrogenase complex and branched-chain alpha-keto acid dehydrogenase complex form multiple species was performed by computer analysis . In addition to 2 previously recognized regions of homology in the alpha subunit, a 3rd region of extensive homology was identified in E1 alpha, and may be one of the sites involved in subunit interaction . E1 beta contains 4 regions of extensive homology . Region 1 contains 10 amino acids that are homologous to a 10-amino acid stretch in Escherichia coli E1 . Regions 2 and 3 have sequence homologies with other dehydrogenases suggesting that these regions may be involved in catalysis. FEBS Lett, 1991 Apr 22, 282(1), 23 - 5 Evidence for the involvement of type II domains in collagen binding by 72 kDa type IV procollagenase; Banyai L et al.; The fibronectin-related region of the 72 kDa type IV procollagenase has been expressed in E . coli as a beta-galactosidase fusion product . The fragment containing the three type II units of the protein was found to have affinity for denatured collagen, suggesting that these domains may be responsible for the collagen-affinity of type IV collagenase . We have also shown that segment Ala-Ala-His-Glu of type IV collagenase (residues 372-375), which is similar to a fibronectin-segment previously implicated in collagen-binding, is not essential for binding activity. Proc R Soc Lond B Biol Sci, 1991 Apr 22, 244(1309), 1 - 9 A comparison of intramolecular rearrangements promoted by transposons Tn5 and Tn10; Ahmed A; The bacterial transposon Tn10 has previously been shown to move to other genomic sites by a conservative mechanism, whereby the transposon is excised by double-strand breaks and inserted between a pair of staggered nicks at the target . Other transposons, like Tn3, have been shown to transpose by a replicative mechanism that involves symmetrical nicking of the element and formation of the 'Shapiro intermediate', which can mature into either a cointegrate or a simple insert . The situation with respect to Tn5 is unclear; it was originally reported to use a conservative mechanism, but other evidence suggests that the mechanism might be replicative . In this paper, rearrangements of adjacent DNA promoted by Tn10 and Tn5 have been compared using positive selection for galactose-resistance to detect such rearrangements . Tn10 promoted the formation of adjacent deletions (that started from an inside end of Tn10), deletion/inversions and simple IS10 insertions, but no cointegrates . This behaviour is fully consistent with a conservative mechanism . In contrast, Tn5 was found to promote formation of adjacent deletions (that started mainly from an outside end of Tn5), IS50 insertions (that were frequently accompanied by inversions of adjacent DNA) and cointegrates . These characteristics seem compatible with a replicative, rather than a conservative, mode of transposition . Clearly, Tn5 and Tn10 exhibit some significant differences in their transposition . These results, and results of some previous experiments, have been interpreted to mean that Tn5 could use a replicative mechanism for its transposition. J Mol Biol, 1991 Apr 20, 218(4), 761 - 7 Functional role of the distal valine (E11) residue of alpha subunits in human haemoglobin; Tame J et al.; We have expressed human alpha-globin to a high level in Escherichia coli as a fusion protein, purified it and removed the N-terminal leader sequence by site-specific proteolysis with blood coagulation factor Xa . The apo globin has been refolded and reconstituted with haem and native beta-globin to form fully functional haemoglobin (Hb) with properties identical to those of native human Hb . By site-directed mutagenesis we have altered the distal residues of the alpha subunits and compared the functional properties of these mutant proteins . The rates of various ligands binding to these proteins in the R-state have been reported by Mathews et al . Here, we present the oxygen equilibrium curves of three E11 alpha mutants and the crystal structures of two of these mutants in the deoxy form . Replacing the distal valine residue of alpha-globin with alanine, leucine or isoleucine has no effect on the oxygen affinity of the protein in either quaternary state, in contrast to the equivalent mutations of beta subunits . The crystal structure of the valine E11 alpha----isoleucine mutant shows that the larger E11 residue excludes water from the haem pocket, but causes no significant movement of other amino acid residues . We conclude that the distal valine residue of alpha-globin does not control the oxygen affinity of the protein by sterically hindering ligand binding. J Mol Biol, 1991 Apr 20, 218(4), 825 - 35 Chimaeric myosin regulatory light chains: sub-domain switching experiments to analyse the function of the N-terminal EF hand; Messer N et al.; The regulatory light chains (RLCs) located on the myosin head, regulate the interaction of myosin with actin in response to either Ca2+ or phosphorylation signals . The RLCs belong to a family of calcium binding proteins and are composed of four "EF hand" ancestral calcium binding motifs (numbered I to IV) . To determine the role of the first EF hand (EF hand I) in the regulatory process, chimaeric light chains were constructed by protein engineering, by switching this region between smooth muscle and skeletal muscle myosin RLCs . For example, chimaera G(I)S consisted of EF hand I of the smooth muscle (gizzard) RLC and EF hands II to IV of the skeletal muscle RLC, whereas chimaera S(I)G consisted of EF hand I of the skeletal muscle RLC and EF hands II to IV of the smooth muscle RLC . The chimaeric RLCs were expressed in Escherichia coli using the pLcII expression system, and after isolation and purification their regulatory properties were compared with those of wild-type smooth and skeletal muscle myosin RLCs . The chimaeric RLCs bound to the myosin heads in scallop striated muscle myofibrils from which the endogenous RLCs had been removed ("desensitized" myofibrils) with similar affinities to those of the wild-type smooth and skeletal muscle RLCs . Both chimaeric RLCs were able to regulate the actin-activated Mg(2+)-ATPase activity of scallop myosin: G(I)S inhibited the ATPase in the presence and absence of Ca2+, like the wild-type skeletal muscle RLC, while S(I)G inhibited the myosin ATPase in the absence of Ca2+, and this inhibition was relieved on Ca2+ addition, in the same way as the wild-type smooth muscle RLC . Thus the type of regulation that the RLCs confer on the myosin is determined by the source of EF hands II to IV rather than that of EF hand I. Cell, 1991 Apr 19, 65(2), 241 - 8 HIV-1 structural gene expression requires the binding of multiple Rev monomers to the viral RRE: implications for HIV-1 latency; Malim MH et al.; Expression of the structural proteins of HIV-1 requires the direct interaction of the viral Rev trans-activator with its cis-acting RNA target sequence, the Rev response element or RRE . Here, we demonstrate that this specific RNA-binding event is, as expected, mediated by the conserved arginine-rich motif of Rev . However, we also show that amino acid residues located proximal to this basic domain that are critical for in vivo Rev function are dispensable for sequence-specific binding to the RRE . Instead, these sequences are required for the multimerization of Rev on the viral RRE target sequence . The observation that Rev function requires the sequential binding of multiple Rev molecules to the RRE provides a biochemical explanation for the observed threshold effect for Rev function in vivo and suggests a molecular model for the high incidence of latent infection by HIV-1. Biochim Biophys Acta, 1991 Apr 17, 1092(2), 153 - 60 High efficiency gene transfection by electroporation using a radio-frequency electric field; Chang DC et al.; In order to develop a safe and effective way to introduce exogenous genes into cells, we have experimented with a new method of electroporation which uses a radio-frequency (RF) electric field to permeabilize the cell membrane . This RF method has several advantages over the conventional electroporation method which uses a direct current (DC) field . We have shown that the RF electroporation method can be used to introduce marker genes into a wide variety of cell lines, including COS-M6, CV-1, CHO, 3T3 and hepatocytes, and is able to increase substantially the efficiency of gene transfection . (For example, the amount of DNA required for transfecting two million COS-M6 cells can be as low as 0.1 microgram) . The transfection efficiency is shown to be affected by a number of factors, including cell type, field strength, pulse protocol and medium buffer . Because of its wide range of applications, high transfection efficiency and lack of harmful side-effect, the RF electroporation method would be particularly useful for introducing genes into human cells for gene therapy. Biochemistry, 1991 Apr 16, 30(15), 3763 - 70 Alternative binding modes for chloramphenicol and 1-substituted chloramphenicol analogues revealed by site-directed mutagenesis and X-ray crystallography of chloramphenicol acetyltransferase; Murray IA et al.; Leucine-160 of chloramphenicol acetyltransferase (CAT) has been replaced by site-directed mutagenesis to investigate enzyme-ligand interactions at the 1-hydroxyl substituent of the substrate chloramphenicol . The consequences of the substitution of Leu-160 by glutamine and by phenylalanine were deduced from the steady-state kinetic parameters for acetyl transfer from acetyl-CoA to the 3-hydroxyl of chloramphenicol and its analogues 1-deoxychloramphenicol and 1-acetylchloramphenicol . The acetyl group of the latter, which is a substrate both in vivo and in vitro, could potentially bind in a similar position to the 1-hydroxyl of chloramphenicol, in close proximity to the side chain of Leu-160 . In the case of Gln-160 CAT, large increases in Km for the three acetyl acceptors were accompanied by small decreases in kcat and in apparent affinity for acetyl-CoA . Such results are consistent with the introduction of the relatively hydrophilic amide in place of the delta-methyl groups of Leu-160 . The kinetic properties of Phe-160 CAT were unexpected in that Km for each of the three acetyl acceptors was unchanged or reduced, compared to the equivalent parameters for the wild-type enzyme, whereas kcat fell significantly (44-83-fold) in each case . The ratios of specificity constants (kcat/Km) for the acetylation of chloramphenicol compared with the alternative acyl acceptors were similar for wild-type and mutant enzymes . As the residue substitutions for Leu-160 do not result in enhanced discrimination against the binding and acetylation of 1-acetylchloramphenicol, it appears unlikely that the 1-acetyl group binds to the CAT active site in the same position as that occupied by the 1-hydroxyl of chloramphenicol.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1991 Apr 16, 30(15), 3612 - 20 Autonomous folding and coenzyme binding of the excised pyridoxal 5'-phosphate binding domain of aspartate aminotransferase from Escherichia coli; Herold M et al.; The coenzyme (PLP) binding domain (residues 47-329) of the dimeric aspartate aminotransferase from Escherichia coli was produced separately by recombinant DNA methods . It folded autonomously both in vivo and in vitro, that is, independently of the native N- and C-terminal extensions that combine to form the small domain of eAAT . The PLP-domain had one binding site for PLP of relatively high affinity involving a covalent bond to the protein . It was monomeric, although the major subunit-subunit interface at the 2-fold symmetry axis remained unchanged . This effect appears to be due mainly to the absence of the N-terminal extension that contains hydrophobic residues, which interact with the PLP-domain of the second subunit in the wild-type dimer . Judged by circular dichroism, fluorescence, and HPLC gel filtration at increasing concentrations of guanidinium chloride, the PLP-domain underwent a three-state unfolding transition (M' in equilibrium M'* in equilibrium U') involving a compact intermediate M'* . This behavior parallels the unfolding of the dissociated native monomer of cAAT. Biochemistry, 1991 Apr 16, 30(15), 3709 - 15 Interaction of fluorescently labeled dideoxynucleotides with HIV-1 reverse transcriptase; Muller B et al.; Succinylfluorescein-labeled dideoxyTTP has been used as a substrate for reverse transcriptase from HIV-1 . On addition to the 3'-end of a primer molecule, there is a reduction of fluorescence yield of a factor of ca . 4 . Release of a fluorescent DNA/DNA primer/template duplex from its complex with reverse transcriptase results in a reduction of fluorescence by a further factor of 2 . The fluorescent nucleotide is incorporated somewhat less efficiently than 3'-azidoTMP and TMP, which show similar incorporation kinetics . Fluorescent chain-terminated primers have been used to investigate the interaction of normal and chain-terminated primer/template complexes with reverse transcriptase . The dissociation constant of a 36/18-mer was 0.65 nM, whereas that of the same complex after the addition of the fluorescent chain-terminating nucleotide to the primer was 3 nM at 25 degrees C . The rate of dissociation of the latter complex from the enzyme was 0.04 s-1 . This was decreased by a factor of ca . 10 at high concentrations (greater than 200 microM) of the nucleotide triphosphate complementary to the next position of the template . The results obtained suggest that potent inhibition of reverse transcriptase activity in in vitro assays results from formation of a slowly dissociating complex between the enzyme and chain-terminated primer/template complexes . However, arguments are presented that lead to the conclusion that this is not the mode of inhibition in cells invaded by HIV . At the prevailing relative concentrations in this situation, chain termination resulting in incomplete transcription is likely to be the major factor. FEMS Microbiol Lett, 1991 Apr 15, 63(2-3), 211 - 7 Variability in LPS composition, antigenicity and reactogenicity of phase variants of Bordetella pertussis; Ray A et al.; Comparison of lipopolysaccharides (LPS) from phase variants of different strains of Bordetella phase variants of different strains of Bordetella pertussis has shown a difference in their composition, antigenicity and reactogenicity . Phase I variants of B . pertussis, with the exception of strain 134, contain a preponderance of LPS I whereas the major component of LPS of phase IV variants is LPS II . Sera raised to LPSs of phase I strains, other than 134, cross-react with each other but not with phase IV LPSs; and similarly all sera raised to phase IV LPSs cross-react with each other and with LPS from 134 phase I . The LPSs of all phase I variants, including that of 134, are approximately ten-fold or more reactive in the limulus amoebocyte lysate assay (LAL) than phase IV LPSs . In the human mononuclear cell pyrogen assay phase IV LPSs also stimulated a lower response than phase I LPSs . The B . pertussis phase I LPSs are 10-times more reactive than Escherichia coli standard endotoxin in the LAL assay but 100-times less reactive than E . coli LPS in the monocyte test for pyrogen . The SDS-PAGE profiles of B . pertussis LPSs are quite different from those of B . parapertussis and B . bronchiseptica strains . B . pertussis LPSs produced a typical lipo-oligosaccharide (LOS) pattern . B . bronchiseptica LPS produced a similar pattern but was antigenically distinct from B . pertussis LPSs I and II . B . parapertussis in contrast produced a ladder pattern typical of smooth type LPS. Biochem J, 1991 Apr 15, 275 ( Pt 2), 447 - 52 Studies on the mechanism of hydroxymethylbilane synthase concerning the role of arginine residues in substrate binding; Lander M et al.; The role of conserved arginine residues in hydroxymethylbilane synthase was investigated by replacing these residues in the enzyme from Escherichia coli with leucine residues by using site-directed mutagenesis . The kinetic parameters for these mutant enzymes and studies on the formation of intermediate enzyme-substrate complexes indicate that several of these arginine residues are involved in binding the carboxylate side chains of the pyrromethane cofactor and the growing oligopyrrole chain. Biochem J, 1991 Apr 15, 275 ( Pt 2), 389 - 91 Homologues of insulinase, a new superfamily of metalloendopeptidases; Rawlings ND et al.; On the basis of a statistical analysis of an alignment of the amino acid sequences, a new superfamily of metalloendopeptidases is proposed, consisting of human insulinase, Escherichia coli protease III and mitochondrial processing endopeptidases from Saccharomyces and Neurospora . These enzymes do not contain the 'HEXXH' consensus sequence found in all previously recognized zinc metalloendopeptidases. Biochem J, 1991 Apr 15, 275 ( Pt 2), 349 - 53 Comparison of the activities of protein disulphide-isomerase and thioredoxin in catalysing disulphide isomerization in a protein substrate; Hawkins HC et al.; 1 . The activities of protein disulphide-isomerase (PDI) and thioredoxin in catalysing disulphide bond isomerization in a protein substrate were compared by using the standard assay, namely the re-activation of 'scrambled' RNAase . 2 . The specific activity of PDI was 25-fold greater than that of thioredoxin . 3 . The greater efficiency of PDI compared with thioredoxin is considered to be due more to the presence of multiple catalytic domains in PDI than to differences in their active-site sequences . 4 . Data and procedures were defined for expressing enzyme activity in standard units, i.e . mumol of active RNAase generated/min. Biochim Biophys Acta, 1991 Apr 15, 1096(3), 245 - 52 Novel muteins of human tumor necrosis factor alpha; Ito R et al.; For chemical synthesis of a gene coding for human tumor necrosis factor alpha (TNF-alpha), DNA sequence predicted by the amino acid sequence of human TNF molecule was prepared . Codons were chosen according to the codon usage in Escherichia coli (E . coli) . The 490 bp gene was assembled by enzymic ligation of 42 oligonucleotides and was cloned into a vector (pKK223-3) for high expression of active TNF-alpha in E . coli . With use of site-directed mutagenesis on this DNA, five different muteins of TNF-alpha were synthesized . TNF-M1 and TNF-M4 have deletions of His-73 and Gln-102, respectively . These deletions didn't cause loss of the cytotoxic activity against L929 cells . TNF-M5, which has a substitution of Asp-10 to Arg, had the similar cytotoxic activity to that of TNF-alpha . The cytotoxic spectra against several tumor cells were not changed by this substitution . TNF-M3 has an amino acid substitution of Glu-116 to His which occupies this position in human TNF-beta . This substitution didn't change the cytotoxicity . In addition, evidence was presented that the change of the carboxyl terminal residue doesn't always influence the cytotoxic activity of TNF-alpha . Many different muteins were also isolated by random mutagenesis with hydroxylamine-HCl . One of the muteins, which carries a mutation of His-15 to Tyr, lost the cytotoxic activity almost completely. Biochem Biophys Res Commun, 1991 Apr 15, 176(1), 517 - 21 Characterization of the inhibition of Escherichia coli pyruvate dehydrogenase complex by pyruvate; Datta A; The E . coli pyruvate dehydrogenase complex was inhibited by pyruvate in absence of its cofactor, NAD+ . The inhibition was found to increase with pH and phosphate concentration of the buffer and decrease with its ionic strength . The inhibition profile was different with MOPS buffer . No radioactivity was found in the enzyme, when the latter was incubated with 2-14C-pyruvate . The results suggest that covalent adduct formation is not necessary for the observed inhibition. Biochem Biophys Res Commun, 1991 Apr 15, 176(1), 447 - 52 Gravidin, an endogenous inhibitor of phospholipase A2 activity, is a secretory component of IgA; Wilson T et al.; Gravidin, a phospholipase inhibitor characterised previously from amniotic fluid, was partially sequenced at the N-terminal and found to be identical to secretory component of human IgA . Inhibition of antiphospholipase activity was observed after incubation of gravidin with monoclonal antibody to human secretory component . Secretory component isolated from human saliva and breast milk was found to inhibit arachidonic acid release from human lymphocytes . It was concluded that gravidin is secretory component of IgA. J Immunol, 1991 Apr 15, 146(8), 2776 - 82 Immunologic and structural characterization of the dominant 66- to 73-kDa antigens of Borrelia burgdorferi; Luft BJ et al.; The 66- to 73-kDa proteins of Borrelia burgdorferi are dominant immunogens and expressed in all strains of B . burgdorferi . The humoral response to these Ag occurs relatively early during the course of infection . Two-dimensional Western blot analysis of this group of Ag revealed them to consist of a tetrad of proteins with apparent molecular mass of 66, 68, 71, and 73 kDa . Furthermore, in this study we demonstrate the 66-kDa protein to be a potent inducer of lymphoproliferation in the patient immune to B . burgdorferi . Monospecific polyclonal antibodies and mAb demonstrate that each of these proteins was immunologically distinct . However, direct amino acid sequence of the 66- and 68-kDa Ag was almost identical and had a high level of sequence similarity to the GroEL heat-shock protein (Hsp60) of Escherichia coli and the 60-kDa immunodominant protein of Treponema pallidum . The amino terminal sequence of the 71- and 73-kDa proteins of B . burgdorferi was almost identical and these proteins had remarkable sequence similarity to the DnaK heat-shock protein of E . coli (Hsp70) . It appears likely, therefore, that proteins related to the heat-shock family are potent immunogens of B . burgdorferi. J Immunol, 1991 Apr 15, 146(8), 2530 - 5 A monoclonal antibody to the human leukocyte adhesion molecule intercellular adhesion molecule-2 . Cellular distribution and molecular characterization of the antigen; Nortamo P et al.; The DNA of the human leukocyte adhesion molecule intercellular adhesion molecule-2 (ICAM-2) was synthesized by the polymerase chain reaction, and a protein A-ICAM-2 fusion protein was expressed in Escherichia coli . The fusion protein was used as immunogen to obtain mAb to ICAM-2 . The 6D5 antibody was selected by its reactivity in immunofluorescence with the endothelial cell line Eahy926 . The antibody precipitated a 55,000 m.w . glycoprotein from radioactivity surface labeled cells and also reacted in Western blotting . As measured by immunofluorescence flow cytometry, the antibody reacted with lymphoblastoid B cells of normal origin, some Burkitt lymphoma cell lines, vascular endothelial cells, the endothelial cell line Eahy926, 4 and a subpopulation of Con A-stimulated blood mononuclear cells . The two Ig-like domains of ICAM-2 were separately expressed in E . coli, and the antibody was shown to react with the NH2-terminal domain . The antibody inhibited the CD11/CD18-dependent binding of HL-60 promyelocytic leukemia cells to transfected COS-1 cells. J Biol Chem, 1991 Apr 15, 266(11), 7207 - 13 Molecular characterization of bovine brain P75, a high affinity binding protein for the regulatory subunit of cAMP-dependent protein kinase II beta; Bregman DB et al.; In mammalian brain, physiological signals carried by cAMP seem to be targeted to intraneuronal sites by the association of cAMP-dependent protein kinase II beta with anchoring proteins that bind the regulatory subunit (RII beta) of the enzyme . Previously, an RII beta-binding domain was characterized in a large (Mr approximately 150,000) candidate anchor protein, rat brain P150 (Bregman, D . B., Bhattacharyya, N., and Rubin, C . S . (1989) J . Biol . Chem . 264, 4648-4656) . RII beta-binding proteins with Mr values of 65,000-80,000 were detected in the brains of other species . Since little was known about the structural features of these lower Mr proteins, we undertook the characterization of bovine brain P75 as a prototype . A cDNA encoding 258 amino acid residues at the C terminus of P75 was cloned by probing a lambda gt11 expression library with 32P-RII beta . The cDNA insert was ligated into the pET-3b expression plasmid, and large amounts of the partial P75 polypeptide (designated P47) were produced in Escherichia coli . A purification scheme that yielded 9 mg of soluble P47 from a 1-liter bacterial culture was devised . Antibodies directed against the P47 polypeptide revealed that P75 is expressed almost exclusively in brain . The sequence of 117 amino acid residues at the C terminus of P75 contains the RII beta-binding site and is 80% identical to the corresponding region of P150 . In contrast, a lower level of identity (36%) between P75 and P150 at a more N-terminal region indicates that the two RII beta-binding proteins are related, but distinct proteins . P75 is not homologous to microtubule-associated protein 2, an RII alpha-selective binding protein, or any other previously studied proteins . C-terminal truncation analysis disclosed that the final 26 residues in P75 are essential for binding RII beta. J Biol Chem, 1991 Apr 15, 266(11), 7081 - 6 Structure-function mapping of interleukin 1 precursors . Cleavage leads to a conformational change in the mature protein; Hazuda DJ et al.; The two interleukin 1 (IL-1) genes (IL-1 alpha and beta) encode 31-kDa precursor molecules, which are cleaved upon secretion to generate the mature, active, carboxyl-terminal 17-kDa proteins . The IL-1 beta precursor is inactive, whereas the IL-1 alpha precursor is as active as the mature IL-1 alpha . In this report, we demonstrate that when either of the recombinant precursors is processed to the mature form, the mature region undergoes a conformational change from a proteinase K-sensitive structure to one that is proteinase K-insensitive . In addition, cysteine residues that are exposed to solvent in the IL-1 beta precursor become buried in the mature protein . Limited structure-activity mapping of the IL-1 beta precursor indicates that the amino-terminal 76 residues are responsible for the conformational change, whereas the most dramatic change in biological activity occurs after further removal of residues 77-94 . These findings suggest that the altered structure of the mature region in precursor IL-1s has been conserved for some function . Denaturation/renaturation experiments implicate the precursor domain in protein folding, and by analogy with signal-directed secretory proteins, the unique conformation of the precursors may play a role in IL-1 secretion. J Biol Chem, 1991 Apr 15, 266(11), 7058 - 66 Site-directed mutagenesis of the RecA protein of Escherichia coli . Tyrosine 264 is required for efficient ATP hydrolysis and strand exchange but not for LexA repressor inactivation; Freitag NE et al.; The role of Tyr264 in nucleotide binding and hydrolysis catalyzed by the RecA protein of Escherichia coli was investigated by constructing Gly, Ser, and Phe substitution mutations using oligonucleotide-directed mutagenesis . The corresponding mutant recA genes neither restored resistance to killing by ultraviolet irradiation nor increased homologous recombination in a recA strain . The purified RecA(Gly264) protein was unable to bind nucleotide, hydrolyze ATP, or form stable ternary complexes with adenosine 5'-O-thiotriphosphate and DNA although the mutant protein bound DNA normally in the absence of nucleotide . The RecA (Phe264) and RecA(Ser264) proteins hydrolyzed ATP poorly and the rates were reduced approximately 8- and 18-fold, respectively . Although capable of low levels of ATP hydrolysis, neither the RecA(Phe264) nor the RecA(Ser264) protein promoted DNA pairing or strand exchange reactions in vitro . Furthermore, these mutant RecA proteins were impaired in their ability to form salt-resistant ternary complexes with adenosine 5'-O-thiotriphosphate) and DNA as judged by filter binding . Nevertheless, nucleoprotein complexes formed with either RecA(Phe264) or RecA(Ser264) protein directed efficient cleavage of LexA repressor in vitro . These results demonstrate that Tyr264 is required for efficient ATP hydrolysis and for homologous pairing of DNA but does not participate in activating RecA protein for LexA repressor autodigestion. J Biol Chem, 1991 Apr 15, 266(11), 7025 - 9 Identification and molecular cloning of yeast homolog of nucleosome assembly protein I which facilitates nucleosome assembly in vitro; Ishimi Y et al.; Yeast DNA coding for nucleosome assembly protein I (NAP-I), which facilitates nucleosome assembly in vitro at physiological ionic conditions, was cloned and its gene product was characterized . A monoclonal antibody against NAP-I (58 kDa) from human HeLa cells was used to screen a genomic library of Saccharomyces cerevisiae constructed into lambda gt11 . A 60-kDa protein was detected by immunoblotting in the extracts of Escherichia coli lysogenized with a positive clone . The 60-kDa protein purified from the extracts had an activity equivalent to that of NAP-I from mouse and human cells . The amino acid sequence deduced from the gene coding for the yeast NAP-I defines a polypeptide of molecular mass 47,848 Da with three negatively charged regions . While the two regions contain 8 and 10 acidic amino acids out of 13 amino acid residues, the longest stretch has 15 glutamic and 13 aspartic acids out of 38 residues . These regions are probably involved in the interaction with histones . Proteins recognized by the anti-NAP-I antibody were also present in Xenopus oocytes and Drosophila cultured cells . Possible roles of NAP-I are discussed in relation to other nucleosome assembly proteins. J Biol Chem, 1991 Apr 15, 266(11), 6999 - 7007 Effects of alternate RNA splicing on glucokinase isoform activities in the pancreatic islet, liver, and pituitary; Liang Y et al.; Different glucokinase isoforms are produced by tissue-specific alternative RNA splicing in the liver and pancreatic islet, the only tissues in which glucokinase activity has been detected . To determine whether differences in protein structure brought about by alternative RNA splicing have an effect on glucose phosphorylating activity, we expressed cDNAs encoding four different hepatic and islet glucokinase isoforms and determined the Km and Vmax of each . When the glucokinase B1 and L1 isoforms were expressed in eukaryotic cells, both high Km glucose phosphorylating activity and immunoreactive protein were detected . However, when the glucokinase B2 and L2 isoforms were expressed, both of which differ by deletion of 17 amino acids in a region between the putative glucose and ATP-binding domains, no high Km glucose phosphorylating activity and much less immunoreactive protein were detected . When the glucokinase B1 and B2 isoforms were expressed in Escherichia coli as fusion proteins with glutathione S-transferase, affinity-purified B1 fusion protein was able to phosphorylate glucose whereas the B2 fusion protein was not, thus indicating that the lack of glucose phosphorylating activity from both the B2 and L2 isoforms is due to lack of intrinsic activity in addition to accumulation of less protein . The Km values of the B1 and L1 isoforms, which differ from each other by 15 amino acids at the NH2 terminus, were similar, but the Vmax of the B1 isoform was 2.8-fold higher than that of the L1 isoform . Mutagenesis of the first two potential initiation codons in the glucokinase B1 cDNA from ATG to GTC (methionine to valine) indicated that the first ATG was crucial for activity and is, therefore, the likely translation initiation codon . Messenger RNAs encoding both the B2 and L2 isoforms of glucokinase were detected in islet and liver by polymerase chain reaction amplification of total cDNA, indicating that mRNAs utilizing this weak alternate splice acceptor site in the fourth exon are normally present in both the liver and islet but as minor components . A regulatory role for weak alternate splice acceptor and donor sites in the glucokinase gene was suggested by examining the expression of the gene in the pituitary and in AtT-20 cells . Interestingly, although glucokinase mRNAs of appropriate sizes were detected in both the AtT-20 cells and rat pituitaries, neither exhibited any detectable high Km glucose phosphorylating activity.(ABSTRACT TRUNCATED AT 400 WORDS) J Biol Chem, 1991 Apr 15, 266(11), 6690 - 2 31phospho-NMR demonstration of phosphocysteine as a catalytic intermediate on the Escherichia coli phosphotransferase system EIIMtl; Pas HH et al.; The mannitol-specific phosphotransferase system transport protein, Enzyme IIMtl, contains two catalytically important phosphorylated amino acid residues, both present on the cytoplasmic part of the enzyme . Recently, this portion has been subcloned, purified, and shown to be an enzymatically active domain . The N-terminal half has also been subcloned and shown to be the mannitol-binding domain . When combined the two domains catalyze mannitol phosphorylation at the expense of phospho-HPr (van Weeghel, R . P., Meyer, G . H., Pas, H . H., Keck, W . H., and Robillard, G . T., Biochemistry in press) . The phospho-NMR spectrum of the purified phosphorylated cytoplasmic domain, taken at pH 8.0, shows two signals, one at -6.9 ppm compared with inorganic phosphate resulting from phosphohistidine and one at +11.9 ppm originating from phosphocysteine . Addition of mannitol plus membranes containing the N-terminal mannitol-binding domain results in the formation of mannitol 1-phosphate and the disappearance of the two signals at -6.9 and +11.9 ppm. J Biol Chem, 1991 Apr 15, 266(11), 6667 - 9 GC-rich DNA sequences block homologous recombination in vitro; Gruss A et al.; The capacity of the RecA protein of Escherichia coli to promote an essential step in homologous recombination, strand transfer, was tested on DNA substrates varying in percentage GC . GC content was determined by a novel method using the polymerase chain reaction . Strand transfer activity is greatly reduced as a function of increasing GC content of the DNA . Some reduction is observed with substrates having a GC percentage similar to that of E . coli . The transfer reaction between sequences adjacent, but not distal, to GC-rich sequences is similarly decreased, suggesting that the structure of RecA-DNA complexes may differ with the GC content of DNA . Our results implicate an important role of DNA sequence in homologous recombination and suggest that many sequences are excluded due to their GC content. Proc Natl Acad Sci U S A, 1991 Apr 15, 88(8), 3479 - 83 Secondary structure of the phosphocarrier protein IIIGlc, a signal-transducing protein from Escherichia coli, determined by heteronuclear three-dimensional NMR spectroscopy; Pelton JG et al.; IIIGlc is a signal-transducing phosphocarrier protein of the phosphoenolpyruvate:glycose phosphotransferase system of Escherichia coli . The secondary structure of IIIGlc is determined by heteronuclear (15N, 13C) three-dimensional NMR spectroscopy . Sequential, medium-range, and long-range nuclear Overhauser effects seen in NMR spectra are used to elucidate 11 antiparallel beta-strands and four helical segments . The medium-range nuclear Overhauser effect patterns suggest that the helices are either distorted alpha-helices or are of the 3(10) class . The amino acids separating the active-site histidine residues (His75 and His90) form two strands (Ala76-Ser81 and Val85-Phe91) of a six-stranded antiparallel beta-sheet that brings His90 and His75 in close proximity . Sequence similarities in IIIGlc and several other sugar-transport proteins suggest that the histidine residues within these proteins may be arranged in a similar manner . The 18-residue N-terminal peptide that precedes beta-strand Thr19-Ile22 in native IIIGlc is disordered and does not interact with the rest of the protein . Furthermore, removal of the N-terminal heptapeptide by a specific endopeptidase does not affect the structure of the remaining protein, thus explaining the phospho-acceptor activity of modified IIIGlc with the phospho-histidine-containing phosphocarrier protein of this system. Proc Natl Acad Sci U S A, 1991 Apr 15, 88(8), 3367 - 71 RecBCD-dependent joint molecule formation promoted by the Escherichia coli RecA and SSB proteins; Roman LJ et al.; We describe the formation of homologously paired joint molecules in an in vitro reaction that is dependent on the concerted actions of purified RecA and RecBCD proteins and is stimulated by single-stranded DNA-binding protein (SSB) . RecBCD enzyme initiates the process by unwinding the linear double-stranded DNA to produce single-stranded DNA, which is trapped by SSB and RecA . RecA uses this single-stranded DNA to catalyze the invasion of a supercoiled double-stranded DNA molecule, forming a homologously paired joint molecule . At low RecBCD enzyme concentrations, the rate-limiting step is the unwinding of duplex DNA by RecBCD, whereas at higher RecBCD concentrations, the rate-limiting step is RecA-catalyzed strand invasion . The behavior of mutant RecA proteins in this in vitro reaction parallels their in vivo phenotypes, suggesting that this reaction may define biochemical steps that occur during homologous recombination by the RecBCD pathway in vivo. Proc Natl Acad Sci U S A, 1991 Apr 15, 88(8), 3024 - 8 Differential effect of cysteine-to-serine substitutions in metallothionein on cadmium resistance; Chernaik ML et al.; A set of mutant coding sequences for Chinese hamster metallothionein (MT) 2 in which codons for individual cysteines were replaced by serine codons was cloned into a yeast expression system . MT gene expression was placed under control of a constitutive promoter on a multicopy Escherichia coli-yeast shuttle vector . MTs were expressed in a metal-sensitive host that lacks the endogenous MT gene . The expressed MTs conferred increased metal resistance to the yeast host . A sensitive assay for cadmium resistance was developed in which population doubling times were monitored in rich liquid medium supplemented with a sublethal dose of CdCl2 . Measurements on mutants with single cysteine replacements at 12 positions revealed two mutant classes . One class (Cys----Ser at position 5, 13, 19, or 33) did not affect the detoxification capacity of MT . A second class (Cys----Ser at position 7, 15, 26, 29, 44, 48, 50, or 60) conferred to the host markedly less resistance to cadmium . Bridging cysteines were more critical to cadmium resistance . All five bridging cysteine mutants studied (at positions 7, 15, 44, 50, and 60) conferred lower cadmium resistance . In contrast, mutation of four out of seven terminal cysteines (at position 5, 13, 19, or 33) was shown to be inconsequential . Mutations tend to be more detrimental in the alpha domain than in the beta domain in conveying cadmium resistance, suggesting that the contribution of individual cysteine to the detoxification function of MT is site specific. Proc Natl Acad Sci U S A, 1991 Apr 15, 88(8), 2984 - 8 Stable three-stranded DNA made by RecA protein; Rao BJ et al.; When RecA protein, in the form of a nucleoprotein filament containing circular single-stranded DNA (plus strand only), reacts with homologous linear duplex DNA, a directional transfer ensues of a strand from the duplex DNA to the nucleoprotein filament, resulting in the displacement of the linear plus strand in the 5' to 3' direction . The initial homologous synapsis, however, can occur at either end of the duplex DNA, or anywhere in between, and when homology is restricted to different regions of the duplex DNA, the joint molecules that form in each region show striking differences in stability upon deproteinization: distal joints greater than proximal joints much greater than medial joints . In the deproteinized distal joints, which are thermostable, 2000 nucleotide residues of the circular plus strand are resistant to P1 nuclease; both strands of the original duplex DNA remain resistant to P1 nuclease, and the potentially displaceable linear plus strand, which has a 3' homologous end, remains resistant to Escherichia coli exonuclease I . These observations suggest that RecA protein promotes homologous pairing and strand exchange via long three-stranded DNA intermediates and, moreover, that, once formed, such triplex structures in natural DNA are stable even when RecA protein has been removed. Proc Natl Acad Sci U S A, 1991 Apr 15, 88(8), 2969 - 73 Sequential truncation of the lactose permease over a three-amino acid sequence near the carboxyl terminus leads to progressive loss of activity and stability; McKenna E et al.; Previous experiments are consistent with the notion that residues 396-401 (.. . SVFTLS ...) at the carboxyl terminus of the last putative transmembrane helix of the lactose (lac) permease of Escherichia coli are important for protection against proteolytic degradation and suggest that this region of the permease may be necessary for proper folding . Stop codons (TAA) have now been substituted sequentially for amino acid codons 396-401 in the lacY gene, and the termination mutants were expressed from the plasmid pT7-5 . With respect to transport, permease truncated at residue 396 or 397 is completely defective, while molecules truncated at residues 398, 399, 400, and 401, respectively, exhibit 15-25%, 30-40%, 40-45%, and 70-100% of wild-type activity . As judged by pulse-chase experiments with {35S}methionine, wild-type permease or permease truncated at residue 401 is stable, while permease molecules truncated at position 400, 399, 398, 397, or 396 are degraded at increasingly rapid rates . The findings indicate that either the last turn of putative helix XII or the region immediately distal to helix XII is important for proper folding and protection against proteolytic degradation. FEMS Microbiol Lett, 1991 Apr 15, 63(2-3), 239 - 46 Low copy number vectors for expression of fused genes to beta-galactosidase in Escherichia coli; Bingham AH; A series of six expression vectors, pXM184Lac.A, B, C, pXM184Z.A, B, C, based on the low copy plasmid pACYC184 that allow for expression of proteins fused to beta-galactosidase in Escherichia coli is described . A level of 50,000 units of beta-galactosidase is routinely observed and is easily identifiable on protein gels . This paper also reports the tight regulation of expression of the Trc promoter in these vectors using the LacIq repressor. J Biol Chem, 1991 Apr 15, 266(11), 6883 - 7 Cloning, expression, and sequence analysis of the genes for carbon monoxide dehydrogenase of Methanothrix soehngenii; Eggen RI et al.; The cdhA and cdhB genes that code for the large and the small subunits of carbon monoxide dehydrogenase (CDH), respectively, were isolated from a genomic library of Methanothrix soehngenii DNA in Escherichia coli, using polyclonal antibodies raised against purified CDH . After introduction in E . coli or Desulfovibrio vulgaris, the cdh genes appeared to be expressed irrespective of their orientation, yielding immunoreactive proteins of 79 and 19 kDa, corresponding in size to the known subunits of purified CDH . However, no CDH activity could be detected in these heterologous hosts . The cdh genes are preceded by consensus ribosome-binding sites and are arranged in an operon-like structure, with cdhA preceding cdhB . Upstream from this operon, sequences similar to archaeal promoters were identified . The amino acid sequence, deduced from the primary sequence of cdhA, showed homology with ferredoxins and with acyl-CoA oxidase . This is compatible with the proposed functions of CDH. J Biol Chem, 1991 Apr 15, 266(11), 6780 - 5 Transmembrane signal transduction and osmoregulation in Escherichia coli . Functional importance of the periplasmic domain of the membrane-located protein kinase, EnvZ; Tokishita S et al.; The EnvZ protein is presumably a membrane-located osmotic sensor which is involved in expression of the ompF and ompC genes in Escherichia coli . Previously, we developed an in vitro method for analyzing the intact form of the EnvZ protein located in isolated cytoplasmic membranes, and demonstrated that this particular form of the EnvZ protein exhibits the ability not only as to OmpR phosphorylation but also OmpR dephosphorylation . In this study, to gain an insight into the structural and functional importance of the putative periplasmic domain of the EnvZ protein, a set of mutant EnvZ proteins, which lack various portions of the periplasmic domain, were characterized in terms of not only their in vivo osmoregulatory phenotypes but also in vitro EnvZ-OmpR phosphotransfer reactions . It was revealed that these deletion mutant EnvZ proteins are normally incorporated into the cytoplasmic membrane . Cells harboring these mutant EnvZ proteins showed a pleiotropic phenotype, namely, OmpF- Mal- LamB- PhoA-, and produced the OmpC protein constitutively irrespective of the medium osmolarity . It was also suggested that all of these mutant EnvZ proteins were defective in their in vitro OmpR dephosphorylation ability, while their OmpR phosphorylation ability remained unaffected . These results imply the functional importance of the periplasmic domain of the EnvZ protein for modulation of the kinase/phosphatase activity exhibited by the cytoplasmic domain in response to an environmental osmotic stimulus. Cancer Res, 1991 Apr 15, 51(8), 2223 - 8 Similarity of expression of low molecular weight G proteins smg p21A and ras p21 in normal and malignant human tissues; Takayama K et al.; We prepared monoclonal antibodies specific for smg p21A, one of the low molecular weight GTP-binding proteins and possibly a suppressor molecule for ras p21 . Two monoclonal antibodies (T22 and T212) reacted with smg p21A but not with Ki-ras p21, both of which were produced by Escherichia coli . These two clones detected an Mr 21,000 band in one-dimensional immunoblotting of extracts of a human pancreatic cancer cell line which was indistinguishable from a band detected by RASK-3, a monoclonal antibody specific for ras p21 . However, T22 and T212 detected a single spot in two-dimensional immunoblotting that was clearly different from the three spots detected in the same cellular extracts by RASK-3 . A series of normal and malignant human tissues were examined for the expression of smg p21A and ras p21 by immunohistochemical methods utilizing T22 and RASK-3 . In essentially all tissues examined, both normal and malignant, smg p21A and ras p21 were expressed with great similarity . Expression of both molecules in all malignant tissues examined was coincident with that in normal tissues except that gastric cancer showed increased expression of the two molecules in comparison with normal gastric tissue. Biochem Biophys Res Commun, 1991 Apr 15, 176(1), 479 - 85 A region immediately adjacent to the origin of replication of bovine papilloma virus type 1 interacts in vitro with the nuclear matrix; Adom JN et al.; We have investigated the interaction of the 69% transforming fragment of the Bovine Papilloma Virus type 1 (BPV1) with the nuclear matrix from 1361.5 cells (NIH-3T3 cells transformed by a BPV chimeric construct) . In vitro studies performed with end-labelled DNA fragments and nuclear matrices prepared using a high-salt extraction procedure demonstrate the binding of a 672 bp fragment adjacent to the viral origin of replication and containing the plasmid maintenance sequence (PMS-1) . This fragment can be cleaved into two pieces (393 and 279 bp), both interacting equally well with the nuclear matrix . This indicates that a least two regions of the 672 bp DNA fragment are involved in the interaction. Biochem Biophys Res Commun, 1991 Apr 15, 176(1), 137 - 44 Expression of rat liver fructose-1,6-bisphosphatase in Escherichia coli; el-Maghrabi MR et al.; Rat liver fructose-1,6-bisphosphatase was expressed in Escherichia coli using a T7 RNA polymerase-transcribed expression system . Maximum yields of soluble active enzyme were obtained when the bacterial host cell, BL21(DE3), carrying the expression plasmid was grown and transcription induced in LB medium at 37 degrees C . Approximately 20mg of fructose-1,6-bisphosphatase are synthesized per liter of culture after 4hr, of which about 10mg are soluble and enzymatically active . Expressed fructose-1,6-bisphosphatase, purified to homogeneity by substrate elution from a carboxymethyl Sephadex column, was indistinguishable from that purified from rat liver in terms of subunit size and kinetic properties . The in vitro expression of fructose-1,6-bisphosphatase in an heterologous system is a necessary preliminary step for future studies on site-directed mutant enzyme forms. J Biol Chem, 1991 Apr 15, 266(11), 6957 - 65 Assay of metabolic superoxide production in Escherichia coli; Imlay JA et al.; Superoxide production has been measured in subcellular fractions of SOD-deficient Escherichia coli provided with physiological reductants . Although cytosolic enzyme(s) do generate O2-., the larger portion is produced by autoxidation of components of the respiratory electron-transport chain . At 37 degrees C and with pO2, NADH, and NAD+ levels matching those in vivo, respiring membrane vesicles generate 3 O2-./10,000 electrons transferred . This corresponds to intracellular O2- . production, in glucose-fed cells, of 5 microM/s . The high SOD content of normal cells restricts O2- . accumulation to 2.10(-10) M, with a moderate gradient from the membrane to the center of the cell . SOD-deficient mutants achieve a much higher steady-state content of O2-. . Rates of superoxide-mediated inactivation of certain enzymes are sufficiently rapid that even 10(-10) M O2- . imposes a significant oxidative stress. J Biol Chem, 1991 Apr 15, 266(11), 6693 - 9 Evidence that the asparagine 322 mutant of the lactose permease transports protons and lactose with a normal stoichiometry and accumulates lactose against a concentration gradient; Franco PJ et al.; The single asparagine 322 mutant of the lactose permease was made by constructing a hybrid plasmid which contained the amino-terminal coding sequence from the wild-type permease gene and the carboxyl-terminal coding sequence from a previously characterized double mutant permease which contained an asparagine residue at position 322 . Since histidine at position 322 has been postulated to be critically involved with H+ transport and the active accumulation of sugars, the ability of the Asn-322 mutant to couple H+ and sugar transport was carefully examined . Measurements of proton/lactose stoichiometries gave very similar values for the wild-type (0.78) and the Asn-322 strain (0.82) . Moreover, the Asn-322 mutant was able to effectively accumulate lactose against a concentration gradient although the levels of accumulation in the Asn-322 mutant (approximately 5-7-fold) were significantly less than that of the wild-type strain (approximately 30-40-fold) . Overall, these results are inconsistent with the notion that an ionizable histidine residue at position 322 is obligatorily required for H+ transport or the active accumulation of galactosides against a concentration gradient . The ability of the Asn-322 mutant to recognize a variety of sugars was compared with wild-type, Val-177, and Val-177/Asn-322 strains . The Asn-322 mutant exhibited an ability to recognize and transport maltose (an alpha-glucoside) which was significantly better than the wild-type strain but not as good as either the single Val-177 mutant or the double Val-177/Asn-322 mutant . Both the Asn-322 and the Val-177/Asn-322 strain showed a relatively poor recognition for alpha-galactosides (i.e . melibiose), beta-galactosides (lactose and thiodigalactoside), and beta-glucosides (cellobiose) . In contrast, the single Val-177 strain exhibited a normal recognition for these sugars. FEMS Microbiol Lett, 1991 Apr 15, 63(2-3), 121 - 5 Ultraviolet light induction of lambda from dcm host strains alleviates EcoRII restriction of phage; Radnedge L et al.; A new form of restriction alleviation is demonstrated for phage induced by ultraviolet light from dcm strains of Escherichia coli K-12 . EcoRII restriction of the induced phage is alleviated, which is the first report of Type II restriction alleviation . Unlike previously reported restriction alleviation, the increase in phage-plating efficiency is not dependent upon irradiation of the plating host for its induction. Proc Natl Acad Sci U S A, 1991 Apr 15, 88(8), 3029 - 32 Replication deficiencies in priA mutants of Escherichia coli lacking the primosomal replication n' protein; Lee EH et al.; The priA gene of Escherichia coli encodes the protein that initiates assembly of the promosome, the entity essential for the replication of phage phi X174 and ColE1-like plasmids in vitro . We have prepared a null priA mutant to assess its role in vivo in replication of phages, plasmids, and the host chromosome . Extracts of this mutant are inert in the initial conversion of the phi X174 viral strand to the duplex form, confirming the absence of the PriA activity . In vivo, the priA mutant fails to produce phi X174 phage and, remarkably, is unable to maintain plasmids that depend on the E . coli chromosome origin as well as those of ColE1 . Deficiencies in cell growth and cell division are also manifest. Cancer Res, 1991 Apr 15, 51(8), 2077 - 83 Inhibition of tumor growth in mice by an analogue of platelet factor 4 that lacks affinity for heparin and retains potent angiostatic activity; Maione TE et al.; An analogue of human platelet factor 4 (PF4) lacking affinity for heparin was specifically designed to evaluate the importance of this property in the antitumor effects of recombinant PF4 . The purified protein, recombinant PF4-241 (rPF4-241), failed to bind heparin but retained the ability to suppress the growth of tumors in mice . Daily intralesional injections of rPF4-241 significantly inhibited the growth of the B-16 melanoma in syngeneic mice without direct inhibitory effects on B-16 cell growth in vitro . Similar antitumor effects were observed with the human colon carcinoma, HCT-116, grown in nude mice, indicating that the inhibitory activity was neither tumor-type specific nor T-cell dependent . rPF4-241 inhibited endothelial cell proliferation in vitro with dose dependence similar to the native sequence rPF4 . Both rPF4 and rPF4-241 inhibited angiogenesis in the chicken chorioallantoic membrane . The analogue, however, was inhibitory at lower concentrations than rPF4 in the chorioallantoic membrane system and its inhibitory effects were not abrogated by the presence of heparin . The present findings support the conclusion that both rPF4 and rPF4-241 inhibit tumor growth by suppression of tumor-induced neovascularization . The finding that this activity is independent of heparin binding may allow the development of PF4-based angiostatic agents with reduced toxicity and improved bioavailability . These results also suggest that PF4 may play a more specific role in modulation of blood vessel development than previously recognized. FEMS Microbiol Lett, 1991 Apr 15, 63(2-3), 233 - 8 Analysis of the variability of S-fimbriae expression in an Escherichia coli pathogen; Ott M et al.; The uropathogenic Escherichia coli wild-type strain 536 produces S-fimbriae, P-related fimbriae and type I fimbriae . Using immuno-colony dot and ELISA techniques, variants were detected showing an increased degree of S-fimbrial production . It was demonstrated by immunofluorescence microscopy that in normal (wild-type) and hyper-S-fimbriated E . coli populations non-fimbriated cells also exist, and that the percentage of S-fimbriated and non-fimbriated bacteria was roughly identical in either population . Hyper-S-fimbriated variants could be stably maintained . The transition from wild-type to hyper-S-fimbriation, which occurs spontaneously, is markedly higher than vice versa . Southern blot analysis of the S fimbrial adhesin (sfa) determinants of normal and hyper-fimbriated strains revealed no marked difference in the gene structure. FEMS Microbiol Lett, 1991 Apr 15, 63(2-3), 219 - 23 The regulation of porin expression in Escherichia coli: effect of turgor stress; Graeme-Cook KA; The OmpF and OmpC porins are major outer membrane proteins of Escherichia coli . Their expression is affected by medium osmolarity such that OmpF is normally produced at low osmolarity and OmpC at high osmolarity . Potassium ion accumulation is a major means by which cells maintain their internal osmolarity in high osmolarity medium in the absence of organic osmolytes such as glycine-betaine . Starvation for potassium causes cells to become turgor stressed . The effect of turgor stress and potassium ion concentration on OmpF and OmpC expression was examined . It was found that ompF gene expression was switched off by turgor stress but there was no concomitant increase in OmpC . Instead, ompC expression responded to the accumulation of potassium ions by the cell in high osmolarity medium. J Gen Microbiol, 1991 Apr, 137 ( Pt 4), 983 - 9 Ammonium transport in Escherichia coli: localization and nucleotide sequence of the amtA gene; Fabiny JM et al.; Escherichia coli expresses a concentrative ammonium (methylammonium) transport system which is strongly repressed under conditions of nitrogen excess . We have previously reported the cloning of a structural gene (amtA) for this transporter by complementation . In this study, a 3.4 kb HindIII-BamHI fragment containing amtA was cloned into the pBluescript KS(+) vector, and unidirectional nested deletions from each end of this 3.4 kb fragment were generated by exonuclease III digestion . The deletions were analysed by complementation of the structural gene mutation produced by Tn10 insertion . This allowed amtA to be localized within a 1.4 kb region which spans the site of the mutation . By application of the Sanger dideoxy method, we sequenced the region containing amtA . The gene contains an open reading frame which encodes a protein with a predicted molecular mass of 27 kDa . The open reading frame is preceded by a putative Shine-Dalgarno sequence and followed by an inverted repeat which might function as a simple transcription terminator . Hydropathic analysis of the inferred amino acid sequence of the gene product predicts that amtA encodes a cytoplasmic component of the ammonium transport system. Infect Immun, 1991 Apr, 59(4), 1521 - 8 Molecular cloning and characterization of a 35.5-kilodalton lipoprotein of Treponema pallidum; Hubbard CL et al.; A clone expressing a 35.5-kDa recombinant treponemal protein was isolated from a genomic DNA library constructed from Treponema pallidum street strain 14 . Polyclonal antiserum raised against the recombinant protein reacted with a corresponding native protein of comparable size in T . pallidum that is specific to the pathogenic treponemes . Radiolabeling of the recombinant protein with {3H}palmitate demonstrated that it is lipid modified . Like other recently characterized T . pallidum lipoproteins, the 35.5-kDa lipoprotein partitioned into the detergent phase from T . pallidum cells fractionated with Triton X-114, suggesting that it is an integral membrane protein . Processing of the recombinant 35.5-kDa lipoprotein from a precursor form to a smaller mature form was not evident in pulse-chase experiments . However, pretreatment of Escherichia coli cells expressing the 35.5-kDa lipoprotein with inhibitors of protein processing or translocation revealed the existence of a higher-molecular-mass precursor . Gene fusion studies with the transposon TnphoA demonstrated the presence of an export signal in the 35.5-kDa lipoprotein that promotes the extracytoplasmic localization of a 35.5-kDa lipoprotein-PhoA hybrid. Nucleic Acids Res, 1991 Apr 11, 19(7), 1687 - 93 Analysis of sequences in the INO1 promoter that are involved in its regulation by phospholipid precursors; Lopes JM et al.; The promoter region of the highly regulated INO1 structural gene of yeast has been investigated . The major transcription initiation start site (+1) was mapped to a position located five nucleotides upstream of the previously identified initiation codon . The INO1 TATA is located at -116 to -111 . The INO1 promoter region was used to construct fusions to the Escherichia coli lacZ gene . All INO1 fusion constructs that retained regulation in response to the phospholipid precursors inositol and choline, contained at least one copy of a nine bp repeated element (consensus, 5'-ATGTG-AAAT-3') . The smallest fragment of the INO1 promoter found to activate and regulate transcription of the fusion gene from a heterologous TATA element was 40 nucleotides in length . This fragment contained one copy of the nine bp repeat and spanned the INO1 promoter region from -259 to -219 . However, when an oligonucleotide containing the nine bp repeated sequence was inserted 5' to the CYC1 TATA element, it failed to activate transcription. Nucleic Acids Res, 1991 Apr 11, 19(7), 1661 - 9 The involvement of transcriptional read-through from internal promoters in the expression of a novel endoglucanase gene FSendA, from Fibrobacter succinogenes AR1; Cavicchioli R et al.; Two distinct mRNA transcripts were synthesized in Escherichia coli during expression of FSendA, an endoglucanase gene from Fibrobacter succinogenes AR1 . Expression of FSendA required a ribosomal frameshift between open reading frame 1 (ORF1) and ORF2 to allow contiguous translation of a 453 amino acid protein (1) . The primary transcript initiated upstream of ORF1 and the secondary transcript from within ORF1 . Both transcripts terminated downstream of ORF2 and termination was essential for endoglucanase expression . Deletion of the primary transcript promoter region allowed read-through of the secondary transcript beyond the terminator region, indicating that a component of the intact FSendA gene allowed efficient transcription termination . The possibility of autogenous regulation by translation products is suggested. Nucleic Acids Res, 1991 Apr 11, 19(7), 1549 - 55 The DNA binding properties of the MutL protein isolated from Escherichia coli; Bende SM et al.; The mutL gene of Escherichia coli, which is involved in the repair of mispaired and unpaired nucleotides in DNA, has been independently cloned and the gene product purified . In addition to restoring methyl-directed DNA repair in extracts prepared from mutL strains, the purified MutL protein binds to both double and single stranded DNA . The affinity constant of MutL for unmethylated single stranded DNA was twice that of its affinity constant for methylated single stranded DNA and methylated or unmethylated double stranded DNA . The binding of MutL to double stranded DNA was not affected by the pattern of DNA methylation or the presence of a MutHLS-repairable lesion. Nucleic Acids Res, 1991 Apr 11, 19(7), 1443 - 7 Effect of uracil situated in the vicinity of a mispair on the directionality of mismatch correction in Escherichia coli; Aprelikova O et al.; We wanted to establish whether strand breaks and gaps, arising during the removal of uracil from newly-synthesized DNA, can be utilized as strand discrimination signals by the methyl-directed mismatch repair system of Escherichia coli . For this purpose, we constructed a series of M13 heteroduplexes that contained a single uracil residue situated either upstream or downstream from a G/T or an A/C mispair . Transfections of these constructs into E . coli strains, either proficient of deficient in mismatch or uracil repair, allowed us to follow the fate of these mispairs in vivo . Our data show that the intermediates of uracil repair cannot substitute for the strand-discrimination signals generated by the MutH protein, which is thought to initiate the methyl-directed mismatch repair process by nicking the unmethylated strand of a newly-synthesized DNA duplex at d(GATC) sites . However, processing of uracil residues situated upstream from the mispair was shown to reduce the yield of the progeny phage arising from the uracil-containing strand, presumably as a result of co-repair of the base analogue and the mispair. Nucleic Acids Res, 1991 Apr 11, 19(7), 1431 - 6 The KUP gene, located on human chromosome 14, encodes a protein with two distant zinc fingers; Chardin P et al.; We have isolated a human cDNA (kup), encoding a new protein with two distantly spaced zinc fingers of the C2H2 type . This gene is highly conserved in mammals and is expressed mainly in hematopoietic cells and testis . Its expression was not higher in the various transformed cells tested than in the normal corresponding tissues . The kup gene is located in region q23-q24 of the long arm of human chromosome 14 . The kup protein is 433 a.a . long, has a M.W . close to 50 kD and binds to DNA . Although the structure of the kup protein is unusual, the isolated fingers resemble closely those of the Kruppel family, suggesting that this protein is also a transcription factor . The precise function and DNA motif recognized by the kup protein remain to be determined. Nucleic Acids Res, 1991 Apr 11, 19(7), 1399 - 405 The C-terminal domain of the Escherichia coli DNA gyrase A subunit is a DNA-binding protein; Reece RJ et al.; We have constructed a clone which over-produces a 33 kDa protein representing the C-terminal portion of the Escherichia coli DNA gyrase A subunit . This protein has no enzymic activity of its own, but will form a complex with a 64 kDa protein (representing the N-terminal part of the A subunit) and the gyrase B subunit, that will efficiently catalyse DNA supercoiling . We show that the 33 kDa protein can bind to DNA on its own in a manner which induces positive supercoiling of the DNA . We propose that the 33 kDa protein represents a domain of the gyrase A subunit which is involved in the wrapping of DNA around DNA gyrase. Nucleic Acids Res, 1991 Apr 11, 19(7), 1369 - 74 Identification of protein binding sites in genomic DNA by two-dimensional gel electrophoresis; Boffini A et al.; We describe a simple two-dimensional electrophoresis procedure to identify the recognition sites of DNA-binding proteins within large DNA molecules . Using this approach, we have mapped E . coli IHF (Integration Host Factor) binding sites within phage Lambda (48 kb) and phage Mu (39 kb) DNA . We are also able to visualize IHF binding sites in E . coli chromosomal DNA (4,700 kb) . We present an extension of this technique using direct amplification by PCR of the isolated restriction fragments, which should permit the cloning of a collection of recognition sequences for DNA binding proteins in complex genomes. Nucleic Acids Res, 1991 Apr 11, 19(7), 1671 - 80 Isolation and footprint analysis of the Escherichia coli thr leader paused transcription complex; Yang MT et al.; The E . coli thr operon leader region contains a cluster of transcription pause sites upstream of the attenuator . In this report, we determine the exact sites of pausing and analyze the structure of the ternary complex by footprint techniques . Under synchronized transcription initiation conditions in vitro, three closely-spaced transcription pause sites were identified . These pause sites appeared downstream of the first region of dyad symmetry, which encodes an RNA hairpin in the transcript, and occurred at positions G112, G114 and A116 of the thr leader RNA . The results showed that the half-life of the thr paused complexes at G112 and G114 could be enhanced by limiting the concentration of the nucleoside triphosphate GTP in the transcription reactions . In addition, the half-life of the paused complexes was shown to increase in the presence of NusA protein . The thr leader complex that paused immediately before residues G112 and G114 of the nascent transcript was isolated and its structure was analyzed with enzymatic and chemical cleavage reagents . The footprinting studies using DNase I showed that there were approximately 35 nucleotides on both strands of the DNA that were protected by RNA polymerase from DNase I cleavage . The DNA segment protected by RNA polymerase is approximately 19 nucleotides upstream and 14 nucleotides downstream of the pause sites . The results from hydroxyl radical footprints also showed a similar pattern of protection at the transcription pause sites . However, no significant differences in the footprinting patterns were observed in the presence or absence of NusA protein. Nucleic Acids Res, 1991 Apr 11, 19(7), 1571 - 6 Upstream sequences of rice proliferating cell nuclear antigen (PCNA) gene mediate expression of PCNA-GUS chimeric gene in meristems of transgenic tobacco plants; Kosugi S et al.; The transgenic tobacco plants have been generated that express the E . coli beta-glucuronidase (GUS) gene under control of the promoter from the rice proliferating cell nuclear antigen (PCNA, DNA polymerase auxiliary protein) gene . GUS expression detected in situ by staining with the chromogenic substrate, 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide (X-Gluc), was restricted to meristems in the organs of the transgenic tobacco plants . This expression responded to the phytohormones which promote callus formation . Furthermore, in situ thymidine uptake showed that the GUS expression pattern corresponded well to the active sites of DNA synthesis . Deletion analysis of the 5' upstream sequence confined the GUS expression pattern to a fragment extending 263 bp upstream of the transcription start site of the rice PCNA gene . Thus, we have identified this fragment as a main regulatory element of the rice PCNA gene promoter. Eur J Biochem, 1991 Apr 10, 197(1), 43 - 7 The binding of the ferric uptake regulation protein to a DNA fragment; Saito T et al.; Using proton NMR, we have studied the binding of a DNA fragment in double-stranded form to the ferric uptake regulation protein, Fur . We have also looked at the binding of {Cr(CN)6}3- to Fur with a view to testing whether binding is due to electrostatic interaction between Fur and the negative surface of the DNA . No competition at the DNA binding site was observed . Additionally, we have examined the binding of manganese ions to Fur in the presence of the DNA fragment and go on to discuss the likely way in which the Fur.DNA complex responds to metal-ion binding to Fur. Eur J Biochem, 1991 Apr 10, 197(1), 39 - 42 The histidines of the iron-uptake regulation protein, Fur; Saito T et al.; There are 12 histidine residues/molecule in the iron-uptake regulation protein (Fur) . Here we examine their pH dependence using proton nuclear magnetic resonance spectroscopy . The histidines have widely spread acid dissociation constants but we can not offer a simple explantation for their complicated behaviour. Eur J Biochem, 1991 Apr 10, 197(1), 203 - 7 Studies on RNase T1 mutants affecting enzyme catalysis; Grunert HP et al.; Using an Escherichia coli overproducing strain secreting Aspergillus oryzae RNase T1, we have constructed and characterized mutants where amino acid residues in the catalytic center have been substituted . The mutants are His40----Thr, Glu58----Asp, Glu58----Gln, His92----Ala and His92----Phe . His92----Ala and His92----Phe mutants are inactive . On the basis of their kcat/Km values, the mutants Glu58----Asp and Glu58----Gln show 10% and 7% residual activity, relative to wild-type RNase T1, whereas the His40----Thr mutant shows 2% activity . The effect of amino acid substitutions on the enzymatic activity of RNase T1 lends further support for a mechanism where Glu58 (possibly activated by His40 and His92 act as general base and acid respectively; this is discussed in terms of the known three-dimensional structure of the enzyme. Eur J Biochem, 1991 Apr 10, 197(1), 29 - 38 Some structural features of the iron-uptake regulation protein; Saito T et al.; An extensive proton nuclear magnetic resonance study of the iron-uptake regulation protein (Fur) from Escherichia coli has been made . Considerable difficulties were experienced in the NMR experiments in 1H2O which may be due unfavourable proton exchange rates in the pH range greater than 6.2, where the protein is soluble . Even in 2H2O, the two-dimensional NMR spectra were not easily interpreted due to widely differing line widths, as a result of the protein side-chains having very differing mobilities . Despite these problems, virtually all the 20 aromatic amino acids have been assigned . Small regions of the protein core were assigned by taking advantage of the approximately 20 non-exchanging peptide-NH resonances in 2H2O . Using two-dimensional J-correlated, homonuclear Hartmann-Hahn and NOE spectroscopies, we have been able to give some assignments in which there is considerable confidence for about one third of the amino acids . Taking advantages of two series of probe experiments, using Mn(II) and a spin label, together with longer range NOE data and result from structure predictions and CD data, we have put forward a tentative fold for the protein which is seen to have a relatively rigid series of interior strands and more flexible exterior strands, many of which are likely to be helical . The Mn(II) probe experiments have also allowed us to define the Fe(II) binding site. Eur J Biochem, 1991 Apr 10, 197(1), 247 - 55 Purification and properties of the H(+)-nicotinamide nucleotide transhydrogenase from Rhodobacter capsulatus; Lever TM et al.; 1 . H(+)-transhydrogenase from Rhodobacter capsulatus is an integral membrane protein which, unlike the enzyme from Rhodospirillum rubrum, does not require the presence of a water-soluble component for activity . 2 . The enzyme from Rb . capsulatus was solubilised in Triton X-100 and subjected to ion-exchange, hydroxyapatite and then gel-exclusion column chromatography . SDS/PAGE of the purified enzyme revealed the presence of two polypeptides with apparent Mr 53,000 and 48,000 . Other minor components which were stained on the electrophoresis gels or which were revealed on Western blots exposed to antibodies raised to total membrane proteins, were probably contaminants . 3 . Antibodies raised to the 53-kDa and 48-kDa polypeptides cross-reacted with equivalent polypeptides in Western blots of solubilised membranes from Rb . capsulatus, Rhodobacter sphaeroides and Rhs . rubrum . The significance of this finding is discussed in the context of the hypothesis {Fisher, R.R . & Earle, S.R . (1982) The pyridine nucleotide coenzymes, pp . 279-324, Academic Press, New York} that the soluble component associated with H(+)-transhydrogenase from Rhs . rubrum is an integral part of the catalytic machinery . Antibodies against the 48-kDa and 53-kDa polypeptides of the Rb . capsulatus enzyme cross-reacted with equivalent polypeptides in solubilised membranes of Escherichia coli . 4 . The dependence of the rate of H- transfer by purified H(+)-transhydrogenase on the nucleotide substrate concentrations under steady-state conditions, the effects of inhibition by nucleotide products and the inhibition by 2'-AMP and by 5'-AMP suggest that the reaction proceeds by the random addition of substrates to the enzyme with the formation of a ternary complex . 5 . In conflict with this conclusion, the reduction of acetylpyridine adenine dinucleotide (AcPdAD+) by NADH in the absence of NADP+ by bacterial membranes was earlier taken as evidence for the existence of a reduced enzyme intermediate {Fisher, R.R . & Earle, S.R . (1982) The pyridine nucleotide coenzymes, pp . 279-324, Academic Press, New York} . However, it is shown here that although chromatophore membranes of Rb . capsulatus catalysed the reduction of AcPdAD+ by NADH, the reaction was not associated with the purified H(+)-transhydrogenase . Moreover, in contrast with the true transhydrogenase reaction, the reconstitution of AcPdAD+ reduction by NADH (in the absence of NADP+) in washed membranes of Rhs . rubrum with partially purified transhydrogenase factor, was only additive. FEBS Lett, 1991 Apr 9, 281(1-2), 93 - 6 Nebulin as a giant actin-binding template protein in skeletal muscle sarcomere . Interaction of actin and cloned human nebulin fragments; Jin JP et al.; Nebulin is a family of giant sarcomere matrix proteins of 600-900 kDa in most vertebrate skeletal muscles . Recent sequence analysis suggests that human nebulin is mainly composed of a large number (greater than 200) of conserved repeats of approximately 35 residues . Two cloned nebulin fragments, consisting of 6 and 8 of the repeats, have been expressed in E . coli using the pET3d vector . Both F-actin cosedimentation and solid-phase binding assays demonstrated a specific binding of these nebulin fragments to actin . This finding suggests that nebulin is a giant protein which binds actin at multiple sites in a template-manner . The presence of an actin-binding template protein in the skeletal muscle sarcomere may have significant implications in the assembly and function of the contractile apparatus. FEBS Lett, 1991 Apr 9, 281(1-2), 67 - 72 Proteolytic activity of plum pox virus-tobacco etch virus chimeric NIa proteases; Garcia JA et al.; Plasmids encoding chimeric NIa-type proteases made of sequences from the potyviruses plum pox virus (PPV) and tobacco etch virus (TEV) have been constructed . Their proteolytic activity on the large nuclear inclusion protein (NIb)-capsid protein (CP) junction of each virus was assayed in Escherichia coli cells . The amino half of the protease seemed to be involved neither in the enzymatic catalysis nor in substrate recognition . In spite of the large homology among the PPV and TEV NIa-type proteases, the exchange of fragments from the carboxyl halves of the molecules usually caused a drastic decrease in the enzymatic activity . Inactive chimeric proteases did not interfere with cleavage by PPV wild type protease expressed from a second plasmid . The results suggest that the recognition and catalytic sites of the NIa proteases are closely interlinked and, although residues relevant for the correct interaction with the substrate could be present in other parts of the protein, a main determinant for substrate specificity should lie in a region situated, approximately, between positions 30 and 90 from the carboxyl end . This region includes the conserved His at position 360 of PPV or 355 of TEV, which has been postulated to interact with the Gln at position -1 of the cleavage sites. FEBS Lett, 1991 Apr 9, 281(1-2), 59 - 63 Pyruvate-formate-lyase-deactivase and acetyl-CoA reductase activities of Escherichia coli reside on a polymeric protein particle encoded by adhE; Kessler D et al.; A 4.8 kb DNA-fragment was cloned and sequenced encompassing the structural gene of PFL-deactivase (2.7 kb) and 2 kb of the 5' flanking region that contains the elements for anaerobic induction . A mutant lacking deactivase was shown to require exogenous electron acceptors for anaerobic growth with glucose . This revealed the identity of PFL-deactivase with the alcohol and acetaldehyde dehydrogenases of E . coli . The multienzyme represents a homopolymeric protein (approximately 40 x 96 kDa) requiring Fe2+ for all functions. FEBS Lett, 1991 Apr 9, 281(1-2), 23 - 6 Rabbit skeletal muscle myosin . Unfolded carboxyl-terminus and its role in molecular assembly; Maeda K et al.; We have expressed in E . coli segments of the rod portion of rabbit skeletal fast muscle myosin and compared physical properties of two different species, LMM-30 and LMM-30C' . LMM-30 consists of 263 amino acids including the original C-terminus of myosin heavy chain . LMM-30C' is colinear with LMM-30, but is devoid of 17 residues at the C-terminus . 1H NMR spectroscopy indicates that the C-terminus of LMM-30, but not of LMM-30C' is unfolded and freely mobile . Furthermore, the present results show that the unfolded C-terminus is essential for molecular assembly of LMM-30; at pH 8.0 LMM-30, but not LMM-30C', formed aggregates upon decreasing the ionic strength. FEBS Lett, 1991 Apr 9, 281(1-2), 181 - 4 Sequence-specific binding of the N-terminal three-finger fragment of Xenopus transcription factor IIIA to the internal control region of a 5S RNA gene; Christensen JH et al.; An N-terminal fragment of Xenopus TFIIIA, containing domains 1-3, (TF 3), was expressed in E . coli . High yields of recombinant zinc finger protein was isolated, and its DNA binding activity for the internal control region (ICR) of the Xenopus 5S RNA gene, was demonstrated by band-shift experiments and DNase I footprinting analysis . TF 3 protects 20 bp of ICR against DNase I digestion . The limits of protection are from +77 to +96 on both coding and noncoding strand . This protection pattern is identical to the protection pattern obtained with TFIIIA in the overlapping region, showing that the 3-finger fragment accounts fully for the protein-DNA interactions in TFIIIA-5S RNA gene over this region. Biochemistry, 1991 Apr 9, 30(14), 3464 - 72 Purification and biochemical characterization of recombinant alpha 1-antitrypsin variants expressed in Escherichia coli; Bischoff R et al.; Site-directed variants of alpha 1-antitrypsin (alpha 1AT) expressed in a recombinant strain of Escherichia coli have been isolated with an overall process yield of 50% following tangential flow ultrafiltration, anion-exchange, immobilized metal affinity, and hydrophobic interaction chromatography . The primary structure of the purified variants including the integrity of the N- and C-termini has been verified by electrospray mass spectrometry of the intact molecules (44 kDa) for two of the variants (alpha 1AT Leu-358 and alpha 1AT Ala-357, Arg-358) . Complementary classical peptide mapping and automated amino acid sequencing have verified 75% of the primary sequence of alpha 1AT Ala-357, Arg-358 . Isoelectric focusing in an immobilized pH gradient revealed some microheterogeneity which proved to be reproducible from one purification batch to another . The isolated variants of alpha 1AT did not show any signs of proteolytic degradation during the purification process and proved to be fully active against their target proteases . The described process also allowed the complete removal of endotoxins from the preparations, opening the possibility to evaluate these novel protease inhibitors for their in vivo efficacy in different animal models of human disease. FEBS Lett, 1991 Apr 9, 281(1-2), 235 - 9 Role of glycine-82 as a pivot point during the transition from the inactive to the active form of the yeast Ras2 protein; Kavounis C et al.; Ras proteins bind either GDP or GTP with high affinity . However, only the GTP-bound form of the yeast Ras2 protein is able to stimulate adenylyl cyclase . To identify amino acid residues that play a role in the conversion from the GDP-bound to the GTP-bound state of Ras proteins, we have searched for single amino acid substitutions that selectively affected the binding of one of the two nucleotides . We have found that the replacement of glycine-82 of the Ras2 protein by serine resulted in an increased rate of dissociation of Gpp(NH)p, a nonhydrolysable analog of GTP, while the GDP dissociation rate was not significantly modified . Glycine-82 resides in a region that is highly conserved between the yeast and human proteins . However, this residue is structurally distant from residues that participate in the binding of the nucleotide, as determined from the crystal structure of the human H-ras gene product . Therefore, the ability of the nucleotide binding site to discriminate between GDP and GTP is dependent not only on residues that are spatially close to the nucleotide, but also on distant amino acids . This is in agreement with the role of glycine-82 as a pivot point during the transition from the GDP- to the GTP-bound form of the Ras proteins. FEBS Lett, 1991 Apr 9, 281(1-2), 64 - 6 The chloroplast gene for ribosomal protein CL23 is functional in tobacco; Yokoi F et al.; Chloroplast rpl23 loci potentially coding for a polypeptide homologous to the E . coli L23 ribosomal protein are frame-shifted in spinach and several other plants, indicating that these loci are pseudogenes . In tobacco, rpl23 constitutes a continuous open reading frame of 93 codons and its transcript initiates at least 66 bp upstream from the initiation codon . The N-terminal amino acid sequence of a 13 kDa protein from the 50 S subunit of tobacco chloroplast ribosomes matches that derived from the tobacco rpl23 locus . This shows that rpl23 is a functional gene in tobacco. Biochemistry, 1991 Apr 9, 30(14), 3401 - 6 Proteolysis of the cytochrome d complex with trypsin and chymotrypsin localizes a quinol oxidase domain; Dueweke TJ et al.; The cytochrome d complex is a two-subunit, membrane-bound terminal oxidase in the aerobic respiratory chain of Escherichia coli . The enzyme catalyzes the two-electron oxidation of ubiquinol and the four-electron reduction of oxygen to water . Previous work demonstrated that the site for ubiquinol oxidation was selectively inactivated by limited proteolysis by trypsin, which cleaves at a locus within subunit I . This work is extended to show that a similar phenomenon is observed with limited chymotrypsin proteolysis of the complex . The cleavage patterns are similar whether one uses the purified oxidase in nondenaturing detergent or reconstituted in proteoliposomes or uses spheroplasts of E . coli as the substrate for the proteolysis . Hence, the protease-sensitive locus is periplasmic in the cell . Fragments resulting from proteolysis were characterized by N-terminal sequencing and by immunoblotting with the use of a monoclonal antibody of known epitope within subunit I . The data indicate that inactivation of the ubiquinol oxidase activity results from cleavage at specific residues with a hydrophilic region previously defined as the Q loop . This domain has been already implicated in ubiquinol oxidation by the use of inhibitory monoclonal antibodies . Electrochemical and HPLC analysis of the protease-cleaved oxidase suggests no global changes in either the quaternary or tertiary structure of the enzyme . It is likely that the Q loop is directly involved in forming a portion of the ubiquinol binding site near the periplasmic surface of the membrane. Biochemistry, 1991 Apr 9, 30(14), 3427 - 31 Terbium(III) luminescence study of tyrosine emission from Escherichia coli glutamine synthetase; Lin WY et al.; Radiationless energy transfer from tyrosine to Tb(III) in Escherichia coli glutamine synthetase and its two mutants (W57L and W158S) has been utilized to assess the tyrosine residue(s) responsible for the observed tyrosine emission and to investigate its spatial relationships to the two metal binding sites of GS . The interference from tryptophan fluorescence was removed by chemical modification of the tryptophan residues by N-bromosuccinimide (NBS) . The Tyr-Tb(III) distances measured by using Forster energy-transfer theory were in good agreement among the three enzymes with average distances of 10.7 and 11.2 A from Tyr to the two metal binding sites . The pKa value for the ionization of tyrosine was determined from fluorescence titration experiments to be approximately 10 for both mutant enzymes . The similarities in pKa values and Tyr-Tb(III) distances observed for all three enzymes lead to the conclusion that the same tyrosine residue(s), is (are) most likely responsible for the Tyr emission . According to the crystal structure distances from tyrosine residues to the two metal binding sites of GS, it is believed that Tyr-179 is the main contributor to the observed Tyr emission . The fact that an intense Tyr emission was observed for W57L GS but not for W158S GS indicates that Trp-57 is much more effective than Trp-158 in quenching the Tyr-179 emission probably through a Forster-type energy transfer . Furthermore, modification of Trp-57 by NBS causes no significant increase in Tyr-179 emission while replacement of Trp-57 by leucine does . This may indicate that oxidized Trp-57 is also an effective quencher for Tyr-179 emission. Biochemistry, 1991 Apr 9, 30(14), 3421 - 6 Fluorescent probes for measuring the binding constants and distances between the metal ions bound to Escherichia coli glutamine synthetase; Lin WY et al.; TNS, 2-p-toluidinylnaphthalene-6-sulfonate, has been used as a fluorescent probe to determine the binding constants of metal ions to the two binding sites of Escherichia coli glutamine synthetase (GS) . TNS fluorescence is enhanced dramatically when bound to proteins due to its high quantum yield resulting from its interactions with hydrophobic regions in proteins . The fluorescence energy transfer from a hydrophobic tryptophan residue of GS to TNS has been detected as an excitation band centered at 280 nm . Therefore, TNS is believed to be bound to a hydrophobic site on the GS surface other than the active site and is located near a hydrophobic Trp residue of GS . GS binds lanthanide ions {Ln(III)} more tightly than either Mn(II) or Mg(II), and the binding constants of several lanthanide ions were determined to be in the range (2.1-4.6) x 10(10) and (1.4-3.0) x 10(8) M-1 to the two metal binding sites of GS, respectively . The intermetal distances between the two metal binding sites of GS were also determined by measuring the efficiencies of energy transfer from Tb(III) to other Ln(III) ions . The intermetal distances of Tb(III)-Ho(III) and Tb(III)-Nd(III) were 7.9 and 6.8 A, respectively. Biochemistry, 1991 Apr 9, 30(14), 3406 - 16 Time-resolved fluorescence studies of genetically engineered Escherichia coli glutamine synthetase . Effects of ATP on the tryptophan-57 loop; Atkins WM et al.; Single-tryptophan-containing mutants of low adenylation state Escherichia coli glutamine synthetase (wild type has two tryptophans at positions 57 and 158) have been constructed and studied by multifrequency phase/modulation fluorescence spectroscopy . The W57L mutant (retains tryptophan at residue 158) and the W158S mutant (retains tryptophan at residue 57) are both characterized by heterogeneous exponential decay kinetics . Global analysis indicates that for the Mn-bound form of the enzyme at pH 7.4 the fluorescence of both tryptophans is best described by a sum of three discrete expontials with recovered lifetimes of 4.77, 1.72, and 0.10 ns for Trp-57 and 5.04, 2.28, and 0.13 ns for Trp-158 . The wild-type enzyme also exhibits decay kinetics described by a triple-exponential model with similar lifetime components . The individual tryptophans are distinguishable by the fractional intensities of the resolvable lifetimes . The wild-type and W158S enzymes are dominated by the 5-ns component which provides nearly 60% and 65%, respectively, of the fractional intensity at five wavelengths spanning the emission spectrum . In contrast, the W57L enzyme demonstrates a larger fraction of the 2-ns lifetime species (60%) and only 35% of the longer lifetime component . The substrate ATP induces a shift to approximately 90% of the 5-ns component for the wild-type and W158S enzymes, whereas the W57L protein is essentially unaffected by this ligand . Steady-state quenching studies with iodide indicate that addition of ATP results in a 3.0-3.5-fold decrease in the apparent Stern-Volmer quenching constants for the wild-type and W158S enzymes . Phase/modulation experiments at several iodide concentrations indicate that the median, 2 ns, lifetime component is selectively quenched compared to the 5-ns lifetime component . These results suggest a model where ATP binding results in a shift in the equilibrium distribution of microconformational states populated by Trp-57 . ATP shifts this equilibrium nearly completely to the states exhibiting the long-lifetime component which, based on quenching studies, is less solvent-accessible than the conformational states associated with the other lifetime components. Biochim Biophys Acta, 1991 Apr 8, 1077(2), 209 - 19 Chemical and kinetic mechanisms of aspartate-beta-semialdehyde dehydrogenase from Escherichia coli; Karsten WE et al.; The chemical and kinetic mechanisms of purified aspartate-beta-semialdehyde dehydrogenase from Escherichia coli have been determined . The kinetic mechanism of the enzyme, determined from initial velocity, product and dead end inhibition studies, is a random preferred order sequential mechanism . For the reaction examined in the phosphorylating direction L-aspartate-beta-semialdehyde binds preferentially to the E-NADP-Pi complex, and there is random release of the products L-beta-aspartyl phosphate and NADPH . Substrate inhibition is displayed by both Pi and NADP . Inhibition patterns versus the other substrates suggest that Pi inhibits by binding to the phosphate subsite in the NADP binding site, and the substrate inhibition by NADP results from the formation of a dead end E-beta-aspartyl phosphate-NADP complex . The chemical mechanism of the enzyme has been examined by pH profile and chemical modification studies . The proposed mechanism involves the attack of an active site cysteine sulfhydryl on the carbonyl carbon of aspartate-beta-semialdehyde, with general acid assistance by an enzyme lysine amino group . The resulting thiohemiacetal is oxidized by NADP to a thioester, with subsequent attack by the dianion of enzyme bound phosphate . The collapse of the resulting tetrahedral intermediate leads to the acyl-phosphate product and liberation of the active site cysteine. J Mol Biol, 1991 Apr 5, 218(3), 557 - 68 Structure and evolution of a group of related aminoacyl-tRNA synthetases; Gatti DL et al.; A yeast nuclear gene, designated MSK1, has been selected from a yeast genomic library by transformation of a respiratory deficient mutant impaired in acylation of mitochondrial lysine tRNA . This gene confers a respiratory competent phenotype and restores the mutant's ability to acylate the mitochondrial lysine tRNA . The amino acid sequence of the protein encoded by MSK1 is homologous to yeast cytoplasmic lysyl-tRNA synthetase and to the product of the herC gene, which has recently been suggested to code for the Escherichia coli enzyme . These observations indicate that MSK1 codes for the lysyl-tRNA synthetase of yeast mitochondria . Several regions of high primary sequence conservation have been identified in the bacterial and yeast lysyl-tRNA synthetases . These domains are also present in the aspartyl- and asparaginyl-tRNA synthetases, further confirming the notion that all three present-day enzymes originated from a common ancestral gene . The most conserved domain, located near the carboxyl terminal ends of this group of synthetases is characterized by a cluster of glycines and is also highly homologous to the carboxyl-terminal region of the E . coli ammonia-dependent asparagine synthetase . A catalytic function of the carboxyl terminal domain is indicated by in vitro mutagenesis of the yeast mitochondrial lysyl-tRNA synthetase . Replacement of any one of three glycine residues by alanine and in one case by aspartic acid completely suppresses the activity of the enzymes, as evidenced by the inability of the mutant genes to complement an msk1 mutant, even when present in high copy . Other mutations result in partial loss of activity . Only one glycine replacement affects the stability of the protein in vivo . The observed presence of a homologous domain in asparagine synthetase, which, like the aminoacyl-tRNA synthetases, catalyzes the formation of an aminoacyladenylate, suggests that the glycine-rich sequence is part of a catalytic site involved in binding of ATP and of the aminoacyladenylate intermediate. J Mol Biol, 1991 Apr 5, 218(3), 529 - 42 Supercoiling is essential for the formation and stability of the initiation complex at the divergent malEp and malKp promoters; Richet E et al.; malEp and malKp are divergent and partially overlapping promoters of the Escherichia coli maltose regulon, whose activity depends on the presence of two transcriptional activators . MalT and CRP (cAMP receptor protein) . Their activation involves a common 210 base-pair regulatory region encompassing multiple binding sites for both activators . Using a supercoiled plasmid containing malEp and malKp as template, purified proteins and a single-round transcription assay, we developed an in vitro system in which both promoters behave as in vivo . In this system, malEp and malKp are active only in the presence of both MalT and CRP, and various mutations in the MalT or CRP binding sites affect the promoters in the same way as they do in vivo . We showed that supercoiling plays a crucial role not only for the formation of the initiation complex at malEp and malKp but also for its stability . In addition, dimethylsulphate protection experiments provide evidence that the nucleoprotein complexes formed by CRP and MalT bound to malEp and malKp on supercoiled and relaxed DNA are different . We speculate that one of the roles of supercoiling might be to assist the assembly of a preinitiation complex involving the regulatory region DNA and several molecules of MalT and CRP. Science, 1991 Apr 5, 252(5002), 128 - 33 Low-resolution real-space envelop