Biochem Biophys Res Commun, 1994 May 30, 201(1), 346 - 55 Fur (ferric uptake regulation) protein interaction with target DNA: comparison of gel retardation, footprinting and electron microscopy analyses; Frechon D et al.; Fur-DNA interactions were analyzed within the regulatory regions of aerobactin and hemolysin operons by a combination of biochemical and ultrastructural methods . Cartography of the Fur binding sites, carried out from electron micrographs, agreed with the data obtained by DNase I footprinting . Visualization of the complexes confirmed the specificity and metal-dependence of Fur binding and demonstrated that the protein polymerizes on its binding sites . Such a polymerization could be involved in the repression process of the bacterial regulator. Biochem Biophys Res Commun, 1994 May 30, 201(1), 242 - 7 Use of single-cysteine mutants to probe the location of the disulfide bond in LamB protein from Escherichia coli; Ling R et al.; The two cysteine residues of the LamB protein of Escherichia coli outer membrane have been shown to form an intrasubunit disulfide whose location differs greatly in the two current topology models of the LamB protein . This study probes the location of the disulfide by examining conditions for intersubunit disulfide formation in single-cysteine mutants of LamB protein . Formation of an intersubunit bond in the purified mutant proteins, which resulted in a disulfide-linked dimer, only occurred after heat treatment, suggesting the disulfide is not exposed on the surface in the native protein. FEBS Lett, 1994 May 30, 345(2-3), 229 - 32 Chaperonin GroE and ADP facilitate the folding of various proteins and protect against heat inactivation; Kawata Y et al.; In the presence of ADP, the molecular chaperones GroEL and GroES from Escherichia coli not only facilitated the refolding of various proteins, but also prevented their irreversible heat inactivation in vitro . Without nucleotides the refolding reactions were arrested by GroEL . Addition of GroES and ADP to the reaction mixture initiated the refolding reactions and the enzyme activities were regained efficiently . The presence of GroE (GroEL and GroES) and ADP also protected against heat inactivation of native enzymes at various temperatures . These findings suggest that in the presence of GroES, nucleotide binding is an important event in the mechanism of GroEL-facilitated protein folding. FEBS Lett, 1994 May 30, 345(2-3), 181 - 6 The formation of symmetrical GroEL-GroES complexes in the presence of ATP; Llorca O et al.; The incubation of chaperonins cpn60 (GroEL) and cpn10 (GroES) from E . coli in the presence of Mg-ATP and KCl generates the formation, as revealed by electron microscopy, of GroEL-GroES complexes with a symmetrical shape in which one toroidal GroES oligomer is bound to each end of the tetradecameric GroEL aggregate (1:2 GroEL:GroES oligomer molar ratio) . The symmetrical complexes are not observed in the presence of ADP or the non-hydrolyzable ATP analog, ATP gamma S, where only asymmetrical complexes (1:1 GroEL:GroES oligomer molar ratio) are formed . These results suggest that ATP hydrolysis is required for the formation of symmetrical complexes. Biochem Biophys Res Commun, 1994 May 30, 201(1), 8 - 15 Inhibition of metal-catalyzed oxidation systems by a yeast protector protein in the presence of thioredoxin; Kwon SJ et al.; A protector protein from Saccharomyces cerevisiae specifically prevents the inactivation of enzymes caused by a thiol/Fe3+/O2 metal-catalyzed oxidation system but not by an ascorbate/Fe3+/O2 system . Ascorbate/Fe3+/O2-mediated damage of enzymes could be prevented by the protector protein only in the presence of reduced thiol . We demonstrate that two proteins from yeast, thioredoxin plus another protein having properties similar to that expected to thioredoxin reductase, when presented with NADPH and the yeast protector protein prevented inactivation of E . coli glutamine synthetase by the ascorbate/Fe3+/O2 system . This system also removes hydrogen peroxide effectively . We also demonstrate evidence suggesting that the NADPH-dependent thioredoxin system reactivates protector protein by reversible disulfide-dithiols exchange. Biochem Biophys Res Commun, 1994 May 30, 201(1), 436 - 42 Activation of soluble guanylyl cyclase by a factor other than nitric oxide or carbon monoxide contributes to the vascular hyporeactivity to vasoconstrictor agents in the aorta of rats treated with endotoxin; Wu CC et al.; We have examined the role of soluble guanylyl cyclase and possible mediators of its activation in the vascular hyporeactivity caused by bacterial endotoxin (lipopolysaccharide, LPS) ex vivo . Treatment of rats with E . coli LPS (10 mg/kg, i.v . for 3h) resulted in a significant reduction in the contractions elicited by norepinephrine (NE; 10(-9)-10(-6) M) in endothelium-denuded aortic rings ex vivo . Methylene blue or LY-83583, inhibitors of soluble guanylyl cyclase, completely restored contractions to NE, whereas the nitric oxide synthase (NOS) inhibitor, N omega-nitro-L-arginine methyl ester (L-NAME), caused only a partial restoration . Zinc protoporphyrin-IX, an inhibitor of heme oxygenase, did not enhance NE-induced contraction in rings from LPS-treated rats, indicating that the production of carbon monoxide (CO) does not contribute to this vascular hyporeactivity . Indomethacin, an inhibitor of cyclooxygenase, further suppressed the contractions in rings from LPS-treated rats . These results suggest that hyporesponsiveness to NE caused by LPS is due to the activation of soluble guanylyl cyclase, which is partially mediated by N(O), but not by CO . Moreover, LPS may induce the production of another mediator(s) that activate soluble guanylyl cyclase in the vascular smooth muscle. Gene, 1994 May 27, 143(1), 95 - 100 Structural proteins of mycobacteriophage I3: cloning, expression and sequence analysis of a gene encoding a 70-kDa structural protein; Ramesh GR et al.; The structural proteins of mycobacteriophage I3 have been analysed by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis (SDS-PAGE), radioiodination and immunoblotting . Based on their abundance the 34- and 70-kDa bands appeared to represent the major structural proteins . Successful cloning and expression of the 70-kDa protein-encoding gene of phage I3 in Escherichia coli and its complete nucleotide sequence determination have been accomplished . A second (partial) open reading frame following the stop codon for the 70-kDa protein was also identified within the cloned fragment . The deduced amino-acid sequence of the 70-kDa protein and the codon usage patterns indicated the preponderance of codons, as predicted from the high G+C content of the genomic DNA of phage I3. Gene, 1994 May 27, 143(1), 1 - 12 The dam and dcm strains of Escherichia coli--a review; Palmer BR et al.; The construction of a variety of strains deficient in the methylation of adenine and cytosine residues in DNA by the methyltransferases (MTases) Dam and Dcm has allowed the study of the role of these enzymes in the biology of Escherichia coli . Dam methylation has been shown to play a role in coordinating DNA replication initiation, DNA mismatch repair and the regulation of expression of some genes . The regulation of expression of dam has been found to be complex and influenced by five promoters . A role for Dcm methylation in the cell remains elusive and dcm- cells have no obvious phenotype . dam- and dcm- strains have a range of uses in molecular biology and bacterial genetics, including preparation of DNA for restriction by some restriction endonucleases, for transformation into other bacterial species, nucleotide sequencing and site-directed mutagenesis . A variety of assays are available for rapid detection of both the Dam and Dcm phenotypes . A number of restriction systems in E . coli have been described which recognise foreign DNA methylation, but ignore Dam and Dcm methylation . Here, we describe the most commonly used mutant alleles of dam and dcm and the characteristics of a variety of the strains that carry these genes . A description of several plasmids that carry dam gene constructs is also included. J Mol Biol, 1994 May 27, 239(1), 15 - 24 Analysis of the N-terminal leucine heptad and hexad repeats of sigma 54; Hsieh M et al.; In order to assess the role of leucine repeat motifs within bacterial protein sigma 54, a series of point mutants were introduced into the many leucine residues near the N terminus . Functional assays in vivo showed that the leucine residues that comprise the previously identified heptad repeat motif are selectively important for function . These heptad leucine residues are critical for mRNA production and also for recognition of the -12 promoter element . An internal proline substitution destroys the function of the heptad repeat region, suggesting a possible alpha-helical structure . Mutants with changes in the distal part of this N-terminal region show the interesting property of allowing nearly full levels of open complex formation, while nonetheless reducing the level of mRNA transcripts produced . All of the above-mentioned properties differ from those exhibited by mutating the interdigitated glutamine residues, which were previously found to be closely involved in the DNA melting reaction . The collection of data suggests that the N-terminal region contains overlapping functional motifs, hydrophobic heptad and glutamine-rich, which together appear to constitute the activation domain of sigma 54. J Biol Chem, 1994 May 27, 269(21), 15362 - 70 Efficient anchoring of RNA polymerase in Escherichia coli during coupled transcription-translation of genes encoding integral inner membrane polypeptides; Ma D et al.; While it has been known that supercoiling of the DNA template can be induced by transcription, the mechanism and the efficiency of this process in vivo is not fully understood . We report here that transcription of genes encoding 16 S rRNA, a stable RNA species, or cytoplasmic polypeptides leads to very little or no detectable DNA supercoiling even under the optimum conditions in Escherichia coli . This indicates that hydrodynamic drag on the transcription complex (including RNA polymerase, nascent RNA, ribosomes, and nascent polypeptides) is not sufficient to anchor RNA polymerase during coupled transcription-translation . On the other hand, transcription of membrane-associated genes encoding integral inner membrane or exported periplasmic polypeptides leads to apparent DNA supercoiling . Transcription of genes encoding integral inner membrane polypeptides leads to significantly greater anchoring of RNA polymerase than does transcription of genes encoding periplasmic polypeptides . This may reflect differences in the coupling of transcription-translation with membrane association during expression of these two classes of polypeptides . Evidence is further presented to suggest that the anchoring of RNA polymerase is probably achieved through the interaction of nascent polypeptides with the cytoplasmic surface of the inner membrane during coupled transcription-translation . Moreover, transcriptions of a membrane-associated gene can, under certain circumstances, induce topological anchoring of an RNA polymerase transcribing a neighboring gene that ordinarily is not membrane-associated . Finally, the potential biological consequences of our findings are discussed. J Biol Chem, 1994 May 27, 269(21), 15217 - 22 Cloning and expression of Ole e I, the major allergen from olive tree pollen . Polymorphism analysis and tissue specificity; Villalba M et al.; Ole e I, the major allergen from the olive tree (Olea europaea), is one of the main causes of allergy in Mediterranean countries and some areas of North America . The cloning and sequencing of several cDNAs coding for the olive allergen have been achieved . cDNA has been synthesized from total pollen RNA and amplified by using the polymerase chain reaction . The nucleotide sequence data demonstrate the existence of microheterogeneities in at least 37 positions out of the 145 amino acids of Ole e I, thus explaining the high degree of polymorphism exhibited by the natural protein . One of the sequenced cDNAs encoding a full-length isoform was inserted into the plasmid vector pGEX-2T and overexpressed . The recombinant Ole e I has been produced in Escherichia coli as a fusion protein with glutathione S-transferase of Schistosoma japonicum . This chimeric protein was purified by affinity chromatography on a glutathione-Sepharose 4B column and digested with thrombin to release the recombinant allergen . Both the fusion protein and the recombinant Ole e I were recognized in Western blot analysis by rabbit polyclonal and mouse monoclonal antisera raised against native Ole e I as well as by the IgE of olive pollen-sensitive human sera . This indicates that the recombinant production of individual isoforms may be useful for the improvement of reagents to be used in diagnosis and therapy of IgE-mediated disorders . In addition, Ole e I mRNA has been observed to be pollen-specific as shown in a Northern blot analysis. J Biol Chem, 1994 May 27, 269(21), 15210 - 6 Mutagenesis of cardiac troponin I . Role of the unique NH2-terminal peptide in myofilament activation; Guo X et al.; Phosphorylation of Ser residues in the NH2-terminal extension unique to cardiac troponin I (cTnI) is known to occur through protein kinase A and to alter myofilament Ca2+ activation (Robertson, S . P., Johnson, J . D., Holroyde, M . J., Kranias, E . G., Potter, J . D., and Solaro, R . J . (1982) J . Biol . Chem . 257, 260-263) . Yet, how the NH2-terminal extension may itself affect thin filament Ca2+ signaling is unknown . To approach this question we have used molecular cloning, mutagenesis, and bacterial synthesis of a full-length cTnI and a truncated mutant (cTnI/NH2) missing the 32 amino acids . Using reconstituted preparations we could show no differences between cTnI and cTnI/NH2 either in inhibition of actomyosin ATPase activity, in Ca(2+)-reversible inhibitory activity, or in the relation between pCa and Ca2+ binding to the regulatory site of cTnC at either pH 7.0 or 6.5 . There were also no significant differences at either pH in the pCa-MgATPase activity relation of myofibrils into which the various species of TnI has been exchanged . Our results indicate: 1) that phosphorylation most likely induces a new state of TnI activity rather than altering an intrinsic effect of the NH2-terminal peptide on Ca2+ activation; and 2) that domains outside the NH2-terminal extension are important with regard to differences in effects of acidic pH on Ca2+ activation on cardiac and skeletal myofilaments. J Biol Chem, 1994 May 27, 269(21), 15132 - 9 Structural requirements of substrate DNA for binding to and cleavage by RuvC, a Holliday junction resolvase; Takahagi M et al.; To elucidate the molecular mechanism of the resolution of Holliday junctions by Escherichia coli RuvC protein, we studied biochemical properties of the protein using various synthetic DNA junctions as model substrates . RuvC cleaves not only a four-way junction but also three-way junctions efficiently . The central core of homology in the junction is essential for the substrates to be cleavable by RuvC . Although the divalent cations are essential for the endonuclease activity, RuvC efficiently forms specific complexes with four-way junctions in the absence of the cations, irrespective of the presence of homologous core sequences . By using T7 endonuclease I as a probe, we studied the topology of the substrate junctions used in our study . The results suggest that RuvC cleaves the three-way junctions with homology core when they become four-way conformers . From the present studies, we propose that RuvC initially binds mostly nonproductively to four-way junctions, which does not require divalent metals, and subsequently cleaves the junctions by a mechanism dependent on a divalent cation and a particular topological conformer that is induced by the sequences at the mobile junctions. J Biol Chem, 1994 May 27, 269(21), 15118 - 23 Human hepatitis virus X gene encodes a regulatory domain that represses transactivation of X protein; Murakami S et al.; The human hepatitis B virus (HBV) X gene seems to be essential for establishment of viral infection, and the X gene product, HBx, transactivates virus and host genes through a wide variety of cis-elements, whereas regulation of HBx has not been fully understood . We found that transactivation-negative HBx mutants truncated at the C-terminal portion specifically repressed the HBx transactivation in trans . The ability to trans-repress the HBx transactivation is confined to the N-terminal third of HBx . Transactivation-positive constructs of HBx were divided into two groups by their sensitivity to trans-repression due to the presence of the N-terminal third . Thus the regulatory domain, the N-terminal third, is separated from the transacting domain and responsible for the negative regulations, the trans-repression and sensitivity to X trans-repression . A possible direct association between the HBx regulatory domains was tested by far-Western blotting using purified fused forms of HBx proteins . The regulatory domain was found to associate preferentially with the full HBx or the regulatory domain, but not with the transacting domain . Taken together, it is possible that HBx has a self-regulatory mechanism that avoids excessive HBx transactivation and is important for regulation of X gene expression. J Biol Chem, 1994 May 27, 269(21), 14974 - 81 The mRNA (guanine-7-)methyltransferase domain of the vaccinia virus mRNA capping enzyme . Expression in Escherichia coli and structural and kinetic comparison to the intact capping enzyme; Higman MA et al.; The mRNA (guanine-7-)methyltransferase active site of the heterodimeric vaccinia virus mRNA capping enzyme was previously localized to the carboxyl-terminal third of the large subunit, D1R, associated with the small subunit, D12L (Cong, P., and Shuman, S . (1992) J . Biol . Chem . 267, 16424-16429; Higman, M . A., Bourgeois, N., and Niles, E . G . (1992) J . Biol . Chem . 267, 16430-16437) . A plasmid was constructed which directs the coexpression of the carboxyl terminus of the D1R subunit from amino acids 498 to 844 and the D12L subunit in Escherichia coli . The mRNA (guanine-7-)methyltransferase catalytic activity in the isolated domain was found to be kinetically equivalent to that present in the intact enzyme . Through mobility shift and ultraviolet photolinkage analyses, both domains were shown to bind RNA in a saturable fashion . RNA binding was localized predominantly to the large subunit, but a low level of linkage of RNA to D12L was also observed . A low, but reproducible, level of mRNA (guanine-7-)methyltransferase activity was detected in the isolated D1R498-844 subunit demonstrating that the active site resides solely within the large subunit of the capping enzyme . This activity is enhanced 30- to 50-fold by the association of the D12L subunit. J Biol Chem, 1994 May 27, 269(21), 14885 - 91 In vitro asymmetric binding of the pleiotropic regulatory protein, FruR, to the ace operator controlling glyoxylate shunt enzyme synthesis; Cortay JC et al.; The fruR gene of Escherichia coli, which encodes the regulatory protein FruR, was cloned in the pT7-5 expression vector so as to overproduce a protein tagged with 6 histidine residues . By using a one-step chromatographic procedure, FruR was purified to near-homogeneity . Analysis of the protein under both denaturing and nondenaturing conditions indicated that it is a tetramer with a molecular mass of about 150 kilodaltons . The positions of interference between FruR and the operator of the acetate operon were examined . The number and nature of the nucleotides essential for FruR binding were determined by several different techniques: base methylation with dimethyl sulfate, base removal by formic acid and hydrazine, uracil interference, and hydroxyl radical footprinting . It was observed that FruR asymmetrically binds to a 16-base pair DNA sequence located 170 base pairs upstream from the transcriptional start point of the ace operon. J Biol Chem, 1994 May 27, 269(21), 15318 - 24 Substrate specificity of Fpg protein . Recognition and cleavage of oxidatively damaged DNA; Tchou J et al.; The 8-oxoguanine-DNA glycosylase of Escherichia coli, also known as formamidopyrimidine-DNA glycosylase (Fpg protein), has N-glycosylase and AP-lyase activities . This enzyme repairs oxidative DNA damage by efficiently removing formamidopyrimidine lesions and 8-oxoguanine residues from DNA . Defined oligodeoxynucleotides containing various 8-oxopurines were used to examine the substrate specificity of Fpg protein and to establish the role of functional groups in DNA on damage recognition and catalysis . Binding affinities of Fpg protein were established for duplex oligodeoxynucleotides containing 8-oxo-2'-deoxyguanine, 8-oxo-2'-deoxyadenine, 8-oxo-2'-deoxynebularine, 8-oxo-2'-deoxyinosine, abasic sites, and a ring-open adduct of C8-aminofluorene guanine . The C8 keto group of 8-oxodG:dC presents in the major groove and is correlated with tight binding (Kd = 8.9 nM) . Binding is much weaker when the C8 keto functional group is in the minor groove, as in 8-oxodG:dA (Kd = 340 nM) . Km and Vmax were determined for the cleavage reaction . Specificity constants (Kcat/Km) are consistently higher for oligodeoxynucleotide duplexes containing 8-oxopurines with C6 and C8 keto groups, as in 8-oxodG:dC and 8-oxodI:dC, where Kcat/Km are 9.3 and 18 min-1 nM x 10(-3), respectively . 8-oxodN:dC lacks the C6 keto group; the specificity constant is 0.024 min-1 nM x 10(-3) . Taken together, our data suggest that the C8 keto group of 8-oxodeoxyguanine and the carbonyl moiety of formamidopyrimidine enable Fpg protein to recognize and bind duplex DNA containing these modified bases . An enzyme-catalyzed reaction involving the C6 keto group of the substrate leads to removal of these lesions . A mechanism involving protonation at O-6 of 8-oxoguanine is proposed to account for the N-glycosylase activity of this enzyme. J Biol Chem, 1994 May 27, 269(21), 15223 - 8 Localization of vitronectin binding domain in plasminogen activator inhibitor-1; Lawrence DA et al.; Plasminogen activator inhibitor type 1 (PAI-1) is the rapid physiologic inhibitor of tissue-type plasminogen activator and urokinase-type plasminogen activator (uPA) . In plasma and the extracellular matrix, PAI-1 is associated with the adhesive glycoprotein vitronectin . In order to characterize the PAI-1 structural domain responsible for binding to vitronectin, the segment of the PAI-1 cDNA encoding amino acids 13-147 (nucleotides 248-650) was randomly mutagenized and subcloned into a bacterial expression vector containing the mature PAI-1 coding sequence . Recombinant PAI-1 mutants were expressed in Escherichia coli and bacterial lysates assayed in duplicate for uPA inhibitory activity and vitronectin binding . Of 190 clones screened, six consistently demonstrated decreased vitronectin binding relative to uPA inhibitory activity . DNA sequence analysis of four of these clones identified 10 unique missense mutations, all located between base pairs 298 and 641, with each clone containing between one and four substitutions . Each substitution was expressed independently by site-directed mutagenesis and again analyzed for uPA inhibitory activity and vitronectin binding . Five point mutations that selectively disrupt vitronectin binding were identified . All 5 residues are located on the exterior of the PAI-1 structure . These findings appear to define a complex binding surface that bridges alpha-helices C and E to beta-strand 1A and includes amino acids 55, 109, 110, 116, and 123 . These results suggest that vitronectin binding may stabilize the active conformation of PAI-1 by restricting the movement of beta-sheet A and thereby preventing insertion of the reactive center loop. Nucleic Acids Res, 1994 May 25, 22(10), 1897 - 902 Mechanism of mutation on DNA templates containing synthetic abasic sites: study with a double strand vector; Takeshita M et al.; Mutagenesis at abasic sites was investigated in E.coli and simian kidney (COS) cells using a duplex shuttle vector containing synthetic analogs of deoxyribose on the phosphodiester backbone . Lesions were positioned on opposite strands of the vector . When the tetrahydrofuranyl analog was used as the abasic site, AT or TA pairs (65-80%) were introduced at the site of the bistrand lesion . Mutagenesis occurred in the absence of SOS induction . Single base deletions (> 80%) dominated the mutational spectra for propanyl and ethanyl analogs of abasic sites lacking a ring structure . For all abasic site analogs, a small proportion of G/C and C/G pairs (6-10%) were observed . dAMP was incorporated predominantly opposite tetrahydrofuranyl sites positioned in the single strand region of a gapped duplex vector . We conclude from these studies that abasic sites positioned in a bistrand configuration are highly mutagenic in E.coli and COS cells . Repair DNA synthesis may be involved in this process. Mol Gen Genet, 1994 May 25, 243(4), 477 - 81 Mapping of transcriptional start sites of the cea and cei genes of the ColE7 operon; Soong BW et al.; Two transcriptional start sites were identified 77 and 78 nucleotides upstream of the translation initiation codon of the colicin E7 gene (ceaE7) . The guanosine nucleotide located at the fifth position of the SOS box is probably a universal transcriptional start site of all E group colicins . Major and minor transcripts of the immunity gene (cei) are initiated at the 3' end of the cea gene . Relative to the -10 sequence, CAAAAT, of the major ceiE7 promoter, the corresponding region of the cei gene of other E group colicins has an increased content of guanosine nucleotides . However the -10 sequence of the minor ceiE7 promoter, TATGAT, was found to be conserved in other colicin promoters . The results indicate that the structure of the major promoter of the ceiE7 gene is unique among the E group colicins. Mol Gen Genet, 1994 May 25, 243(4), 409 - 16 The catalytic domain of Escherichia coli K-12 adenylate cyclase as revealed by deletion analysis of the cya gene; Crasnier M et al.; In Escherichia coli, adenylate cyclase activity is regulated by phosphorylated EnzymeIIA(Glc), a component of the phosphotransferase system for glucose transport . In strains deficient in EnzymeIIA(Glc), cAMP levels are very low . Adenylate cyclase containing the D414N substitution produces a low level of cAMP and it has been proposed that D414 may be involved in the process leading to activation by EnzymeIIA(Glc) . In this work, spontaneous secondary mutants producing large amounts of cAMP in strains deficient in EnzymeIIA(Glc) were obtained . The secondary mutations were all deletions located in the cya gene around the D414N mutation, generating adenylate cyclases truncated at the carboxyl end . Among them, a 48 kDa protein (half the size of wild-type adenylate cyclase) was shown to produce ten times more cAMP than wild-type adenylate cyclase in strains deficient in EnzymeIIA(Glc) . In addition, this protein was not regulated in strains grown on glucose and diauxic growth was abolished . This allowed the definition of a catalytic domain that is not regulated by the phosphotransferase system and produces levels of cAMP similar to that of regulated wild-type adenylate cyclase in wild-type strains grown in the absence of glucose . Further analysis allowed the characterization of the COOH-terminal regulatory domain, which is proposed to be inhibitory to the activity of the catalytic domain. Mol Gen Genet, 1994 May 25, 243(4), 400 - 8 ADP-glucose pyrophosphorylase in shrunken-2 and brittle-2 mutants of maize; Giroux MJ et al.; The Shrunken-2 (Sh2) and Brittle-2 (Bt2) genes of maize encode subunits of the tetrameric maize endosperm ADPglucose pyrophosphorylase . However, in all sh2 and bt2 mutants so far examined, measurable ADPglucose pyrophosphorylase activity remains . We have investigated the origin of the residual activity found in various sh2 and bt2 mutants as well as tissue specific expression and post-translational modification of the Sh2 and Bt2 proteins . Sh2 and Bt2 cDNAs were expressed in Escherichia coli and antibodies were prepared against the resulting proteins SH2 and BT2 specific antibodies were used to demonstrate that SH2 and BT2 are endosperm specific, are altered or missing in various sh2 or bt2 mutants, and have a mol . wt . of 54 and 51 kDa respectively in the wild type . The Sh2 and Bt2 transcripts are also endosperm specific . Ten sh2 and eight bt2 mutants show varying severity of phenotypes expressed at transcript, protein subunit and kernel level . Synthesis of multiple transcripts and proteins commonly occurs as a result of sh2 or bt2 mutation . While all mutants produce detectable enzymic activity, not all produce detectable transcripts and proteins . To examine the origin of the apparent non-SH2/BT2 endosperm enzymic activity, homologs of Sh2 and Bt2, designated Agp1 and Agp2 respectively, were isolated from an embryo cDNA library and found to hybridize to endosperm transcripts distinct from those of Sh2 and Bt2 . Thus Agp1 and Agp2 or closely related genes may be responsible for the residual activity in some sh2 and bt2 mutants . Surprisingly, no evidence of post-translational modification of the SH2 and BT2 protein subunits was detected. Mol Gen Genet, 1994 May 25, 243(4), 379 - 89 Requirement of the Pro-Cys-His-Arg sequence for O6-methylguanine-DNA methyltransferase activity revealed by saturation mutagenesis with negative and positive screening; Ihara K et al.; O6-Methylguanine-DNA methyltransferase catalyzes transfer of a methyl group from O6-methylguanine and O4-methylthymine of DNA to a cysteine residue of the enzyme protein, thereby repairing the mutagenic and carcinogenic lesions in a single-step reaction . There are highly conserved amino acid sequences around the methyl-accepting cysteine site in eleven molecular species of methyltransferases . To elucidate the significance of the conserved sequence, amino acid substitutions were introduced by site-directed mutagenesis of the cloned DNA for Escherichia coli Ogt methyltransferase, and the activity and stability of mutant forms of the enzyme were examined . When cysteine-139, to which methyl transfer occurs, was replaced by other amino acids, all of the mutants showed the methyltransferase-negative phenotype . Methyltransferase-positive revertants, isolated from one of the negative mutants, had restored codons for cysteine . Thus the cysteine residue is essential for acceptance of the methyl group and is not replaceable by other amino acids . Using this negative and positive selection procedure, the analysis was extended to other residues near the acceptor site . At the histidine-140 and arginine-141 sites, all the positive revertants isolated carried codons for amino acids identical to those of the wild-type protein . At proline-138, five substitutions (serine, glutamine, threonine, histidine, and alanine) exhibited the positive phenotype but levels of methyltransferase activity in extracts of cells harboring these mutant forms were very low . This suggests that the proline residue at this site is important for maintaining the proper conformation of the protein . With valine-142 substitutions there were seven types of positive revertants, among which mutants carrying isoleucine, cysteine, leucine, and alanine showed relatively high levels of methyltransferase activity . These results indicate that the sequence Pro-Cys-His-Arg is a sine qua non for methyltransferase to exert its function. Mol Gen Genet, 1994 May 25, 243(4), 374 - 8 Regulation of replication of plasmid pBR322 in amino acid-starved Escherichia coli strains; Herman A et al.; The stringent response causes inhibition of replication of plasmid pBR322 in amino acid-starved Escherichia coli cells whereas in relaxed mutants the replication of this plasmid proceeds for several hours . On the basis of density shift experiments and pulse-labelling experiments we showed that most of the pBR322 molecules begin replication during the relaxed response and the rate of plasmid DNA synthesis in unstarved and isoleucine-starved relA- bacteria is similar . We found that the Rom function plays a key role in the stringent control of plasmid pBR322 replication, as insertional inactivation of the rom gene causes amplification of pBR322rom- in both relA- and relA+ strains during amino acid starvation . Moreover, pUC19, which is a pBR322-derived plasmid lacking the rom gene, behaves like pBR322rom-, whereas introduction of the rom gene into the pUC19 replicon drives it into the pBR322 mode of replication in amino acid-starved bacteria . A model for the regulation of pBR322 plasmid DNA replication by Rom protein in amino acid-starved Escherichia coli strains is proposed. Nucleic Acids Res, 1994 May 25, 22(10), 1866 - 73 Removal of 3'-phosphoglycolate from DNA strand-break damage in an oligonucleotide substrate by recombinant human apurinic/apyrimidinic endonuclease 1; Winters TA et al.; A recombinant human AP endonuclease, HAP1, was constructed and characterized with respect to its ability to recognize and act upon a model double-stranded 39-mer oligodeoxyribonucleotide substrate containing a strand break site with 3'-phosphoglycolate and 5'-phosphate end-group chemistries . This oligodeoxyribonucleotide substrate exactly duplicates the chemistry and configuration of a major DNA lesion produced by ionizing radiation . HAP1 was found to recognize the strand break, and catalyze the release of the 3'-phosphoglycolate as free phosphoglycolic acid . The enzyme had a Vmax of 0.1 fmole/min/pg of HAP1 protein, and a Km of 0.05 microM for the 3'-phosphoglycolate strand break lesion . The mechanism of catalysis was hydrolysis of the phosphate ester bond between the 3'-phosphoglycolate moiety and the 3'-carbon of the adjacent dGMP moiety within the oligonucleotide . The resulting DNA contained a 3'-hydroxyl which supported nucleotide incorporation by E . coli DNA polymerase I large fragment . AP endonucleolytic activity of HAP1 was examined using an analogous double-stranded 39-mer oligodeoxyribonucleotide substrate, in which the strand break site was replaced by an apyrimidinic site . The Vmax and Km for the AP endonuclease reaction were 68 fmole/min/pg of HAP1 protein and 0.23 microM, respectively. Mol Gen Genet, 1994 May 25, 243(4), 434 - 41 A ribosomal frameshifting error during translation of the argI mRNA of Escherichia coli; Fu C et al.; Using fusions between the Escherichia coli genes argI and lacZ, it has been demonstrated that ribosomal frameshifting occurs at a frequency of between 3% and 16% within the argI mRNA, soon after the initiation codon . The frameshift involves a phenylalanyl-tRNA shifting into the +1 frame at the sequence UUU-U/C . The shift does not occur if the in-frame phenylalanine codon UUU is replaced by UUC . The level of frameshifting is higher in dense cultures and is not dependent on phenylalanine starvation . In the wild-type argI gene this frameshifting event would be an error, leading to a truncated, non-functional protein . Therefore, it is unlike the numerous examples of required frameshifting events that have been described in other genes. Proc Natl Acad Sci U S A, 1994 May 24, 91(11), 5027 - 9 An algorithm to generate low-resolution protein tertiary structures from knowledge of secondary structure; Monge A et al.; An algorithm is described to assemble the three-dimensional fold of a protein starting from its secondary structure . A reduced representation of the polypeptide chain is used together with a crude potential based on pair hydrophobicities . The method is shown to be successful in locating the native topology for two 4-alpha-helix bundles, myohemerythrin and cytochrome b-562. Proc Natl Acad Sci U S A, 1994 May 24, 91(11), 5017 - 21 Formation of a ternary complex by human XPA, ERCC1, and ERCC4(XPF) excision repair proteins; Park CH et al.; The xeroderma pigmentosum complementation group A (XP-A) protein, XPA, has recently been expressed in Escherichia coli in a soluble and fully functional form . An affinity column was prepared by linking the XPA protein to a solid support . When HeLa cell-free extract capable of excision repair was applied to the column, > 99.9% of the proteins were in the flow-through . However, the flow-through fraction lacked excision activity . The activity was restored by adding the high salt (1 M KCl) eluate of the column to the flow-through fraction . The XPA protein-bound fraction was tested for specific proteins by an in vitro complementation assay with a panel of cell-free extracts from DNA repair-deficient human and rodent cell lines . The XPA-bound fraction complemented cell-free extracts of excision repair cross-complementing 1 (ERCC-1), ERCC-4 (XP-F), and XP-A mutants . We conclude that the XPA damage recognition protein makes a ternary complex with the ERCC1/ERCC4(XPF) heterodimer with a potential nuclease function. Proc Natl Acad Sci U S A, 1994 May 24, 91(11), 4703 - 7 Subunit dynamics in Escherichia coli preprotein translocase; Joly JC et al.; SecY, SecE, and band 1 copurify as the SecY/E integral membrane domain of Escherichia coli preprotein translocase . To measure the in vivo association of these polypeptides and assay possible exchange, plasmid-borne secY and secE genes were placed under control of the ara regulon and fused to DNA encoding the influenza hemagglutinin epitope . Cells were incubated with {35S}methionine, grown for a "chase" period, and then induced with arabinose to express epitope-tagged, nonradioactive SecY and SecE . Both the wild-type and epitope-tagged polypeptides assembled into functional, heterotrimeric SecY/E complex . However, immunoprecipitation with antibody to the epitope tag did not cross-precipitate radiolabeled SecY or SecE . Thus, these subunits normally associate stably in vivo. Biochemistry, 1994 May 24, 33(20), 6356 - 62 Thermodynamic properties of the transition state for the rate-limiting step in the folding of the alpha subunit of tryptophan synthase; Chen X et al.; To gain insight into the physical properties of the transition state for the rate-limiting step in the folding of the alpha subunit of tryptophan synthase from Escherichia coli, the urea dependence of the unfolding reaction was examined as a function of temperature . Consistent with a previous, more limited study {Hurle, M.R., Michelotti, G.A., Crisanti, M.M., & Matthews, C.R . (1987) Proteins 2, 54}, the activation entropy for unfolding was found to be negative above 4 M urea . The present study extends this finding to show that both the activation entropy and enthalpy decrease with increasing urea concentrations between 4 and 7.5 M . The change in the heat capacity from the native to the transition state is positive and appears to increase with the denaturant concentration . The urea and temperature dependences of the unfolding rates were analyzed in terms of the denaturant-binding model of Tanford {Tanford, C . (1970) Adv . Protein Chem . 24, 1} . The values for the activation enthalpy and activation entropy of binding are in good agreement with those obtained from a calorimetric study of urea binding to unfolded proteins {Makhatadze, G.I., & Privalov, P.L . (1992) J . Mol . Biol . 226, 491} . These results show that (1) the binding of urea to the transition state of the alpha subunit has thermodynamic properties which are similar to those for urea binding to unfolded proteins, (2) the transition state is distinct from the unfolded conformation and exposes only a fraction of its urea-binding sites to solvent, and (3) the negative value for the activation entropy for unfolding reflects, in part, the ordering of urea on newly exposed surfaces.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1994 May 24, 33(20), 6350 - 5 Detection of an intermediate in the folding of the (beta alpha)8-barrel N-(5'-phosphoribosyl)anthranilate isomerase from Escherichia coli; Jasanoff A et al.; We have used thermodynamic and kinetic techniques to monitor the guanidinium chloride induced (GdmCl-induced) denaturation of N-(5'-phosphoribosyl)anthranilate isomerase from Escherichia coli (ePRAI) . Although CD-monitored equilibrium denaturation curves are consistent with cooperative unfolding of the protein centered at 1.45 M GdmCl, fluorescence readings drop by over 25% in the region preceding the CD-monitored transition, suggesting non-two-state behavior . Kinetics experiments measure a slow relaxation rate with negative fluorescence amplitude when protein is diluted from 0 to 0.5 M GdmCl, corroborating results from equilibrium conditions . Detection of several unfolding and refolding rates in final GdmCl concentrations from 0 to 5.0 M indicates the presence of at least one intermediate along unfolding and refolding pathways . GdmCl dependence of the relaxation rates can be explained most easily by a nonsequential mechanism for ePRAI unfolding, though a sequential mechanism cannot be ruled out . The data corroborate the fragment complementation studies of Eder and Kirschner {Eder, J., & Kischner, K . (1992) Biochemistry 31, 3617-3625}, which are consistent with unfolding of the C-terminal portion of a yeast-derived PRAI in its folding intermediate . In ePRAI, such partial unfolding would expose W391 to quenching by solvent molecules; W356, ePRAI's other tryptophan, is buried in the hydrophobic core and is unlikely to be affected by local changes in structure . A C-terminally unfolded folding intermediate has been demonstrated in the folding of tryptophan synthase (alpha-subunit), a related beta alpha-barrel enzyme.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1994 May 24, 33(20), 6213 - 20 Modulation of the oxidation-reduction potential of the flavin in lipoamide dehydrogenase from Escherichia coli by alteration of a nearby charged residue, K53R; Maeda-Yorita K et al.; The epsilon-amino group of a lysine residue occupies a position within bonding distance of the flavin N5 and the bound NADPH pyridinium C4' in glutathione reductase, and it has been suggested that this positive charge influences the redox potential of the FAD {Pai & Schulz (1983) J . Biol . Chem . 258, 1752} . A conserved lysine residue occupies a similar position in lipoamide dehydrogenase . This residue has been replaced by an arginine in lipoamide dehydrogenase from Escherichia coli to give K53R . The spectral and redox properties of the FAD in K53R as well as the interaction of the flavin with bound NAD+ are profoundly affected by the change . K53R does not catalyze either the dihydrolipoamide-NAD+ or the NADH-lipoamide reactions except at very low concentrations of the reducing substrate . The absorbance spectrum of K53R in the visible and near-ultraviolet is little changed from that of wild-type enzyme, but in contrast, the spectrum of K53R is sensitive to pH with an apparent pKa = 7.0 . Unlike the wild-type enzyme, the binding of beta-NAD+ to K53R alters the spectrum and indicates an apparent Kd = 7.0 microM at pH 7.6 . The flavin fluorescence is partially quenched, and the visible and near-ultraviolet circular dichroism spectrum is changed by beta-NAD+ . K53R is extensively reduced (mostly EH4) by 2 equiv of dihydrolipoamide/FAD while the wild-type enzyme is only partially reduced (mostly EH2) . The rate of this reduction is lowered by approximately 3-fold relative to the wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1994 May 24, 33(20), 6177 - 85 Mapping critical residues in eukaryotic DNA-binding proteins: a plasmid-based genetic selection strategy with application to the Oct-2 POU motif; Botfield MC et al.; Discrimination between allowed and disallowed amino acid substitutions provides a powerful method for analysis of protein structure and function . Site-directed mutagenesis allows specific hypotheses to be tested, but its systematic application to entire structural motifs is inefficient . This limitation may be overcome by genetic selection, which allows rapid scoring of thousands of randomly (or pseudorandomly) generated mutants . To facilitate structural dissection of DNA-binding proteins, we have designed two generally applicable bacterial selection systems: pPLUS selects for the ability of a protein to bind to a user-defined DNA sequence, whereas pMINUS selects against such binding . Complementary positive and negative selections allow rapid mapping of critical residues . To test and calibrate the systems, we have investigated the bipartite POU domain of the human B-cell-specific transcription factor Oct-2 . (i) An invariant residue (Asn347) in the DNA-recognition helix of the POU-specific homeodomain (POUHD) was substituted by each of the 19 other possible amino acids . The mutant proteins each exhibited decreased specific DNA binding as defined in vivo by genetic selection and in vitro by gel retardation; relative affinities correlate with phenotypes in the positive and negative selection systems . An essential role for Asn347 in wild-type POUHD-DNA recognition is consistent with homologous Asn-adenine interactions in cocrystal structures of canonical homeodomains . (ii) Extension of pPLUS/pMINUS selection to the POU-specific subdomain (POUs) is demonstrated by analysis of mutations in its putative helix-turn-helix (HTH) element.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1994 May 24, 33(20), 6167 - 76 Effects of base composition on the negative cooperativity and binding mode transitions of Escherichia coli SSB-single-stranded DNA complexes; Lohman TM et al.; We have examined the ability of the Escherichia coli single-stranded DNA binding protein (SSB) tetramer to form its different binding modes on poly(dC), poly(U), and poly(A) over a range of NaCl and NaF concentrations for comparison with previous studies with poly(dT) . In reverse titrations with poly(U) and poly(A) at 25 degrees C, pH 8.1, SSB forms all four binding modes previously observed with poly(dT), namely, (SSB)35, (SSB)40, (SSB)56, and (SSB)65, where the subscript denotes the site size (i.e., the average number of nucleotides occluded per SSB tetramer) . As with poly(dT), the low site size modes are favored at low monovalent salt concentration (< 10 mM), whereas increasing salt concentration facilitates the transitions to the higher site size modes . Surprisingly, SSB does not form a stable (SSB)35 complex on poly(dC), even at 1 mM NaCl; rather, the (SSB)56 mode is formed under these conditions . Upon raising the {NaCl}, the (SSB)56 complex undergoes a transition to the (SSB)65 complex (transition midpoint, 40 mM NaCl) . On the basis of studies with dC(pC)34, dT(pT)34, and dA(pA)34, the inability of the SSB tetramer to form the (SSB)35 complex with poly(dC) is due mainly to a much lower degree of negative cooperativity for binding oligodeoxycytidylates to the SSB tetramer . At low salt concentration, the negative cooperativity parameter, sigma 35, is lowest for dA(pA)34, intermediate for dT(pT)34, and highest for dC(pC)34, indicating that it is most difficult to saturate the SSB tetramer with two molecules of dA(pA)34 . We have also measured the equilibrium constants for binding the oligodeoxynucleotides dC(pC)34, dC(pC)69, dA(pA)34, and dA(pA)69 as a function of {NaCl} and {NaBr} and find that the salt dependencies of these oligonucleotides are dependent upon base composition . These studies also indicate that ion binding accompanies formation of these SSB-ss-DNA complexes, although there is a net release of ions upon formation of the complex . This influence of both salt concentration and base composition indicates that both electrostatic and nonelectrostatic factors contribute to the negative cooperativity associated with ss-DNA binding to the SSB tetramer. Biochemistry, 1994 May 24, 33(20), 6100 - 9 Attractant- and disulfide-induced conformational changes in the ligand binding domain of the chemotaxis aspartate receptor: a 19F NMR study; Danielson MA et al.; The isolated ligand binding domain of the chemotaxis aspartate receptor is the focus of the present study, which both (a) identifies structural regions involved in the attractant-induced conformational change and (b) investigates the kinetic parameters of attractant binding . To analyze the attractant-induced conformational change within the homodimeric domain, 19F NMR is used to monitor six para-fluorophenylalanine (4-F-Phe) positions within each identical subunit of the homodimer . The binding one molecule of aspartate to the homodimer perturbs three of the 4-F-Phe resonances significantly: 4-F-Phe150 in the attractant binding site, 4-F-Phe107 located 26 A from the site, and 4-F-Phe180 at a distance of 40 A from the site . Comparison of the frequency shifts triggered by aspartate and glutamate reveals that these attractants generate different conformations in the vicinity of the attractant site but trigger indistinguishable long-range conformational effects at distant positions . This long-range conformational change is specific for attractant binding, since formation of the Cys36-Cys36' disulfide bond or the nonphysiological binding of 1,10-phenanthroline to an aromatic pocket distal to the attractant site each yield conformational changes which are significantly more localized . The attractant-triggered perturbations detected at 4-F-Phe107 and 4-F-Phe180 indicate that the structural change includes an intrasubunit component communicated through the domain to its C-terminal region, which, in the full-length receptor, continues through the membrane as the second membrane-spanning helix . It would thus appear that the transmembrane signal is transmitted through this helix.(ABSTRACT TRUNCATED AT 250 WORDS) Proc Natl Acad Sci U S A, 1994 May 24, 91(11), 5133 - 7 Three-dimensional working model of M1 RNA, the catalytic RNA subunit of ribonuclease P from Escherichia coli; Westhof E et al.; A three-dimensional model of M1 RNA, the catalytic RNA subunit of RNase P from Escherichia coli, was constructed with the aid of a computer . The modeling process took into account data from chemical and enzymatic protection experiments, phylogenetic analysis, studies of the activities of mutants, and the kinetics of reactions catalyzed by the binding of substrate to M1 RNA . The model provides a plausible picture of the binding to M1 RNA of the tRNA domain of a precursor tRNA substrate . The scissile bond and adjacent segments of the aminoacyl acceptor stem of a precursor tRNA substrate can fit into a cleft that leads to the phylogenetically conserved, central part of the structure. Proc Natl Acad Sci U S A, 1994 May 24, 91(11), 5114 - 8 Solution NMR structure of the major cold shock protein (CspA) from Escherichia coli: identification of a binding epitope for DNA; Newkirk K et al.; Sequence-specific 1H and 15N resonance assignments have been determined for the major cold shock protein (CspA) from Escherichia coli with recently developed three-dimensional triple-resonance NMR experiments . By use of these assignments, five antiparallel beta-strands were identified from analysis of NMR data . Strands 1-4 have a classical 3-2-1-4 Greek key beta-sheet topology and there are two beta-bulges, at positions Lys10-Trp11 and Gly65-Asn66 . Three-dimensional structures of CspA were generated from NMR data by using simulated annealing with molecular dynamics . The overall chain fold of CspA is a beta-barrel structure, with a tightly packed hydrophobic core . Two-dimensional isotope-edited pulsed-field gradient 15N-1H heteronuclear single-quantum coherence spectroscopy was used to characterize the 15N-1H fingerprint spectrum with and without a 24-base oligodeoxyribonucleotide, 5'-AACGGTTTGACGTACAGACCATTA-3' . Protein-DNA complex formation perturbs a subset of the amide resonances that are located mostly on one face of the CspA molecule . This portion of the CspA molecular surface includes two putative RNA-binding sequence motifs which contribute to an unusual cluster of eight surface aromatic side chains: Trp11, Phe12, Phe18, Phe20, Phe31, His33, Phe34, and Tyr42 . These surface aromatic groups, and also residues Lys16, Ser44, and Lys60 located on this same face of CspA, are highly conserved in the family of CspA homologues . These isotope-edited pulsed-field gradient NMR data provide a low-resolution mapping of a DNA-binding epitope on CspA. Proc Natl Acad Sci U S A, 1994 May 24, 91(11), 4713 - 7 TAR RNA-binding protein is an inhibitor of the interferon-induced protein kinase PKR; Park H et al.; A cDNA encoding a double-stranded-RNA (dsRNA)-binding protein was isolated by screening a HeLa cell cDNA expression library for proteins that bind the HIV-1 Rev-responsive-element RNA . The cDNA encoded a protein that was identical to TRBP, the previously reported cellular protein that binds the transactivation response element (TAR) RNA of human immunodeficiency virus type 1 . TRBP inhibited phosphorylation of the interferon-induced ribosome-associated protein kinase PKR and of the eukaryotic translation initiation factor eIF-2 alpha in a transient-expression system in which the translation of a reporter gene was inhibited by the localized activation of PKR . TRBP expression in HeLa cells complemented the growth and protein-synthesis defect of a vaccinia virus mutant lacking the expression of the dsRNA-binding protein E3L . These results implicate TRBP as a cellular regulatory protein that binds RNAs containing specific secondary structure(s) to mediate the inhibition of PKR activation and stimulate translation in a localized manner. FEBS Lett, 1994 May 23, 345(1), 23 - 6 Prediction of the structural similarity between spermidine/putrescine-binding protein and maltose-binding protein; Matsuo Y et al.; A structural similarity between spermidine/putrescine-binding protein and maltose-binding protein has been predicted by a sequence-structure compatibility method . The sequence alignment obtained by this method revealed a consensus sequence motif located on the surface loop between the first alpha-helix and the second beta-strand, and the further analysis identified a similar motif in iron-binding protein . The conservation of this motif among certain bacterial periplasmic binding proteins suggests a common functional role for this region as well as an evolutionary relationship between them. J Mol Biol, 1994 May 20, 238(5), 852 - 3 Crystallization and preliminary crystallographic data for an FMN-dependent nitroreductase from Escherichia coli B; Skelly JV et al.; An FMN-dependent nitroreductase enzyme isolated from Escherichia coli B has been crystallized in a form suitable for high-resolution structural analysis . The crystals belong to the tetragonal space group P4(1)2(1)2 or its enantiomorph P4(3)2(1)2 with cell parameters a = b = 57.74 A, c = 275.51 A and two molecules per asymmetric unit . Diffraction extends to beyond 1.9 A. J Mol Biol, 1994 May 20, 238(5), 669 - 81 A thermodynamic study of the trp repressor-operator interaction; Ladbury JE et al.; We have measured the heats of formation of the trp repressor/operator complex by direct titration calorimetry over the temperature range 10 degrees C to 40 degrees C . A primary strong mode of binding displays the characteristic large negative heat capacity change observed by other methods in the formation of specific protein/DNA complexes . Unlike most such reactions, however, the formation of the trp repressor/operator complex is enthalpically driven throughout the physiological temperature range . After saturation of this principal mode, we also detected a secondary weaker binding mode, which we ascribe to a now well documented interaction called "half-site" binding . Although weak, this mode also exhibits an unusually large negative heat capacity change . Since the interface of the proposed secondary half-site binding mode has the same complementary stereochemistry as the primary one (due to internal symmetry), we correlate the negative heat capacity change with the formation of a stereospecific interface and not with high affinity . As in similar cases, the empirical correlation between buried non-polar surfaces and reduction of heat capacity does not account for the large negative delta Cp, nor do crystal structures reveal any further reduction in solvent excluded surfaces within the reactants upon complex formation . We attribute the "unaccounted for" decrement in the heat capacity of the complex to the stereospecific restriction of the hydrated polar elements that form the specific interface . We suggest that the "tightening of soft internal modes" at and near the polar interface of the complex is more important than previously recognized because previous considerations did not take into account the highly hydrated nature of these polar elements and the concomitant reduction in the degrees of freedom of the water structure. J Mol Biol, 1994 May 20, 238(5), 643 - 8 Which nucleotides in the "-10" region are crucial to obtain a fully active MalT-dependent promoter? Danot O, Raibaud O. We have replaced the hexanucleotide corresponding to the "-10" region of malPp, a positively regulated promoter from Escherichia coli, by random nucleotide sequences and isolated 48 different variants that were as active as the wild-type promoter . Analysis of the nucleotide sequence of their "-10" region strongly suggests that the nature of the nucleotide present at three positions plays a crucial role: 46 of the 48 malPp variants contained C or T at position -12, A at position -11 and T at position -7 . The nucleotide composition at the three other positions seems to be much more flexible . The features of these "-10" regions are similar to those of the constitutive promoters, but some significant differences are noticeable and will be discussed . In contrast to other positively regulated promoters, none of the various "-10" regions, including the consensus sequence TATAAT, led to a constitutive promoter. J Biol Chem, 1994 May 20, 269(20), 14620 - 4 Glycoproteins with Gal alpha 4Gal are absent from human erythrocyte membranes, indicating that glycolipids are the sole carriers of blood group P activities; Yang Z et al.; The antigenic determinants of the blood group P family (P1, P, Pk, and LKE antigens) are chemically based on Gal alpha 4Gal . For human erythrocytes it has been claimed that the majority of P1 determinants are expressed in glycoproteins, mainly band 4.5 (Haselberger, C . G., and Schenkel-Brunner, H . (1982) FEBS Lett . 149, 126-128) . In the present work, the existence of Gal alpha 4Gal in glycoproteins of erythrocyte membranes (ghosts) of P1 positive and negative human individuals was carefully analyzed on replicas of sodium dodecyl sulfate-polyacrylamide electrophoresis gels using specific reagents (Escherichia coli HB101/pDC1 expressing the Pap gene and monoclonal antibodies with specificities for P1 and Pk antigens) . No binding to glycoproteins was detected with any of these ligands when the ghosts had been pretreated with butanol to remove glycolipids . Therefore, all antigenic determinants of the P blood group family on human red cells are exclusively expressed in glycolipids and are absent from glycoproteins. Carbohydr Res, 1994 May 20, 258, 233 - 41 Structure of the capsular antigen of Escherichia coli O8:K50:H-; Grue MR et al.; The primary structure of the acidic capsular antigen of Escherichia coli O8:K50:H- was shown by glycose analysis, methylation analysis, and one- and two-dimensional 1H and 13C NMR spectroscopy to be composed of repeating linear tetrasaccharide units having the structure: (formula: see text). J Biol Chem, 1994 May 20, 269(20), 14672 - 80 Comparison of deoxyoligonucleotide and tRNA(Lys-3) as primers in an endogenous human immunodeficiency virus-1 in vitro reverse transcription/template-switching reaction; Arts EJ et al.; We developed an endogenous in vitro reverse transcription assay to study the properties of priming and template switching during human immunodeficiency virus (HIV) replication . Reactions were primed with HIV reverse transcriptase (RT) and either a deoxyoligonucleotide primer (dPR) or tRNA(Lys-3), the natural primer for reverse transcription . The RNA templates utilized were the actual HIV sequences involved in the first template switch, namely a primer binding sequence (PBS)/U5/R RNA donor template and a R/U3 RNA acceptor template . Reverse transcription reactions using the latter templates and dPR or tRNA(Lys-3) as primers yielded four major products: (-)-strong-stop DNA, a partial template-switched DNA, full template-switched DNA, and a pseudo-PBS-primed product . Use of dPR resulted in three times less template switching than was obtained with tRNA(Lys-3) . When reactions were primed with either dPR or tRNA(Lys-3), increases in acceptor:donor template ratios resulted in augmented template switching . Increasing the concentration of RT resulted in increased priming from the PBS but had no effect on the efficiency of template switching . Decreasing the extent of R region overlap resulted in a drop in efficiency of template switching . Decreases in the R region on the donor template also caused a drop in initiation of transcription that was primed by tRNA(Lys-3) from the PBS . In contrast, a corresponding reduction of the R region on the acceptor template had no effect on priming . We conclude that a transcriptional complex of tRNA(Lys-3) and RT may be associated not only with the PBS but also with other cis RNA sequences and secondary structures in a manner essential for efficient priming and template switching. J Biol Chem, 1994 May 20, 269(20), 14566 - 74 Glycogen synthase kinase-3 beta is a dual specificity kinase differentially regulated by tyrosine and serine/threonine phosphorylation; Wang QM et al.; The enzyme glycogen synthase kinase-3 (GSK-3) has been implicated in the control of several metabolic enzymes and transcription factors in response to extracellular signals . In the past, the enzyme has been considered to be a protein Ser/Thr kinase although it was recently reported to contain Tyr(P) (Hughes, K., Nikolakaki, E., Plyte, S . E., Totty, N . F., and Woodgett, J . R . (1993) EMBO J . 12, 803-808) . A cDNA encoding rabbit skeletal muscle GSK-3 beta was cloned and expressed in Escherichia coli as an active protein kinase, with apparent M(r) 46,000, capable of phosphorylating several known GSK-3 substrates . Recombinant GSK-3 beta autophosphorylated on Ser, Thr, and Tyr residues although the enzyme already contained Tyr(P) as judged by its recognition by anti-Tyr(P) antibodies . The net result of the autophosphorylation was a 3-5-fold reduction in enzyme activity . GSK-3 alpha, purified from rabbit muscle, also underwent autophosphorylation but only on Ser and Thr residues . In this case, the autophosphorylation stabilized the enzyme activity compared with the control lacking ATP/Mg2+ . Of several phosphatases tested, the lambda-phage phosphatase was the most effective in dephosphorylating at Ser and Thr residues but did not dephosphorylate at Tyr residues . The action of the lambda-phosphatase caused a reactivation of GSK-3 beta to approximately 80% of the starting activity . The protein tyrosine phosphatase PTP1B was able to dephosphorylate at Tyr residues leading to a reduction in enzyme activity . A truncated form of GSK-3 beta, apparent M(r) 40,000, had a significantly higher specific activity, was defective in autophosphorylation, and was not inactivated in the autophosphorylation reaction . We conclude that GSK-3 beta is a dual specificity protein kinase in the same sense as the mitogen-activated protein kinase/ERK family of enzymes . Phosphorylation at different residues differentially controls enzyme activity, Ser/Thr phosphorylation causing inactivation and Tyr phosphorylation resulting in increased activity. Biochim Biophys Acta, 1994 May 18, 1206(1), 90 - 6 Spectroscopic, electrochemical, and ligand binding properties of the horse heart metmyoglobin His64-Tyr variant; Tang HL et al.; The distal histidine (E7) of horse heart myoglobin (Mb) has been replaced by tyrosine using site-specific mutagenesis . The resulting green Mb variant (His64-Tyr) was expressed in Escherichia coli JM101, isolated and purified to homogeneity . Spectrophotometric pH titrations of the variant exhibit a change in spectrum that occurs with a pKa of 4.7 (25 degrees C) . The midpoint reduction potential of the variant is 20 mV (vs . SHE at pH 7, 25 degrees C) . Cyanide and azide binding measurements indicate that the oxidized variant binds these anionic ligands with much greater affinity at pH 4.0 than at neutral pH . Extended X-ray absorption fine structure (EXAFS) spectroscopy establishes that the variant is six coordinate at pH 7.0 and pH 4.2 . Higher shell contributions to the iron EXAFS observed at pH 7.0 are attributed to tyrosine . These contributions are absent at pH 4.2 . Thus, the sixth heme iron ligand of the oxidized variant Mb at pH 7.0 is attributed to oxygen from the hydroxyl group of tyrosine and the sixth ligand present at pH 4.2 is attributed to the oxygen atom of a coordinated water . The EXAFS spectra, electronic absorption spectra, and ligand binding properties of the His64-Tyr Mb variant are consistent with the binding of Tyr-64 as the sixth heme iron ligand between pH 5 and 12 and with the replacement of Tyr-64 by a water molecule at low pH with a pKa of 4.7. Biochim Biophys Acta, 1994 May 18, 1206(1), 143 - 54 Strong-field and integral spin-ligand complexes of the cytochrome bo quinol oxidase in Escherichia coli membrane preparations; Calhoun MW et al.; The cytochrome bo-type terminal oxidase of Escherichia coli is an analogue of mammalian aa3-type cytochrome c oxidase . The catalytic core of both enzymes is a binuclear site containing a penta-coordinate heme (heme o or a3) and copper (CuB) . Herein we report on UV-visible and magnetic properties of ligand complexes of the binuclear site of cytochrome bo . Cyanide, sulfide, and azide react with the Fe(3+)-Cu+ center to give EPR-detectable low-spin complexes, analogous to those formed by cytochrome aa3 . Analyses of the ligand fields of these complexes indicate that heme o has a single axial histidine ligand . Cyanide and azide react with the Fe(3+)-Cu2+ center to yield forms observable via UV-visible spectroscopy but not EPR . With formate and fluoride, cytochrome bo forms integral spin complexes similar to those of cytochrome aa3 . These complexes have UV-visible characteristics of high-spin species, but EPR spectra show features which appear to correspond to transitions within an integral spin multiplet . Cytochrome bo forms another integral spin complex with azide and NO which is nearly identical to the azide-NO species in cytochrome aa3 . This suggests that the binuclear centers of the two enzymes are quite similar. Biochim Biophys Acta, 1994 May 18, 1206(1), 113 - 9 Catalytic core of rat tyrosine hydroxylase: terminal deletion analysis of bacterially expressed enzyme; Walker SJ et al.; Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in catecholamine biosynthesis . This enzyme is hypothesized to consist of an amino-terminal regulatory domain and a carboxy-terminal catalytic domain . In the present studies, we have utilized recombinant DNA techniques to map the boundaries of the regulatory and catalytic domains of TH . We have isolated a full-length cDNA clone for rat pheochromocytoma TH and have expressed the enzyme in bacteria . Utilizing this bacterial expression system and polymerase chain reaction technology, we have constructed and subcloned genes for five amino-terminal deletion mutants (N delta 40, N delta 155, N delta 165, N delta 175 and N delta 200; N delta denotes amino-terminal deletion and the numerical value denotes the number of amino acids deleted) and two carboxy-terminal deletion mutants (C delta 19 and C delta 50) . The catalytic core of rat tyrosine hydroxylase has been identified to include the region from amino acid #165 to amino acid #479 . The amino-terminal deletion mutants, N delta 40, N delta 155 and N delta 165 are from 1.85 to 2.5-fold more active than unmodified recombinant TH, while the removal of 19 amino acids from the C-terminus (C delta 19) results in a 70% reduction in enzyme activity . Removal of additional sequences (ten more residues from the N-terminus {N delta 175}; or an additional 31 amino acids from the C-terminus {C delta 50}) results in protein that is totally without enzyme activity . As expected, removal of 40 (or more) N-terminal amino acids abolishes the ability of the catalytic subunit of the cAMP-dependent protein kinase to phosphorylate the recombinant enzyme; serine-40 is the phosphorylation site on TH for PKA . We conclude that the N-terminal boundary for the TH catalytic domain resides between residues 165 and 175 and that removal of this N-terminal domain (totally or partially) increases the activity of the enzyme . These findings confirm previous reports that proteolytic cleavage at amino acid #158 produces an active (and activated) catalytic fragment. Biochim Biophys Acta, 1994 May 17, 1218(1), 21 - 6 On the binding of isolated yeast tRNA(Phe) anticodon arm to Escherichia coli 30S and 70S ribosomes . Guanosine-42 is important for the binding; Nekhai SA et al.; Conditions for the reversible binding of isolated anticodon arm of yeast tRNA(Phe) (15-nucleotide, corresponding to nucleotides N28-N42) to the P site of Escherichia coli 30S and 70S ribosomes have been determined . The affinity constant for the anticodon arm binding to 70S ribosomes is shown to be only 30-fold weaker than that for the binding of total tRNA(Phe) . The affinities of yeast tRNA(Phe) and the anticodon arm for 30S subunits and of the anticodon arm for the total 70S ribosomes are shown to be equal . These data imply that the anticodon arm moiety of tRNA(Phe) mainly contributes to the tRNA-70S ribosome interaction, i.e., it contributes for the most part to the total free energy of the deacylated tRNA(Phe) interaction with the P site of the 70S ribosome . By taking into account additional contacts in the 3'-CCA end, other contacts in the region besides the anticodon arm and 3'-CCA end are presumably absent . Within the anticodon arm removal of the 3'-end nucleotide corresponding to guanosine-42 in tRNA(Phe) decreases the association constant of the anticodon arm-ribosome interaction 15-fold . Replacement of this guanosine with other nucleosides as well as modification of the ribose moiety (oxidation and reduction) does not affect the affinity . The replacement data are very likely to indicate that G42 affects the anticodon arm affinity by forming a direct contact with ribosome. Biochim Biophys Acta, 1994 May 17, 1218(1), 119 - 22 Expression and myristoylation of NAP-22 using a baculovirus transfer vector system; Maekawa S et al.; NAP-22 is a brain enriched acidic protein localized in the membrane fraction . The peptide sequence of NAP-22 elucidated from the cDNA sequencing, however, showed that NAP-22 is a very hydrophilic protein having no transmembrane regions . The peptide sequence analysis also showed that NAP-22 has a consensus sequence of N-myristoylation and the presence of polybasic domain in its N-terminal region . These sites could be the anchoring sites to localize to the membrane . In this study, we showed the myristoylation of NAP-22 using a Baculovirus expression system and also showed a liposome binding ability of the expressed protein in Escherichia coli. Biochemistry, 1994 May 17, 33(19), 5974 - 83 Reactivity and ionization of the active site cysteine residues of DsbA, a protein required for disulfide bond formation in vivo; Nelson JW et al.; DsbA is a periplasmic protein of Escherichia coli that appears to be the immediate donor of disulfide bonds to proteins that are secreted . Its active site contains one accessible and one buried cysteine residue, Cys30 and Cys33, respectively, which can form a very unstable disulfide bond between them that is 10(3)-fold more reactive toward thiol groups than normal . The two cysteine residues have normal properties when in a short peptide . In DsbA, the Cys30 thiol group is shown to be reactive toward alkylating reagents down to pH 4 and to be fully ionized, on the basis of the UV absorbance of the thiolate anion at 240 nm . Its reactivity is altered by another, unknown group on the reduced protein titrating with a pKa of about 6.7 . The other cysteine residue is buried and unreactive and has a high pKa value . The ionization properties of the DsbA thiol groups can explain, at least partly, the high reactivity of its disulfide bonds and thiol groups at both neutral and acidic pH values. Biochemistry, 1994 May 17, 33(19), 5942 - 6 Mutagenesis at a highly conserved phenylalanine in cytochrome P450 2E1 affects heme incorporation and catalytic activity; Porter TD; The phenylalanine corresponding to Phe-429 of rabbit cytochrome P450 2E1 is 1 of approximately 10 highly conserved residues in this superfamily of over 200 sequenced enzymes . This nearly invariant residue has been postulated to be involved in electron transfer between the heme of cytochrome P450cam and its redox partners {Stayton, P.S., Poulos, T . L., & Sligar, S.G . (1989) Biochemistry 28, 8201-8205} . To test this hypothesis, oligonucleotide-directed mutagenesis was used to replace this amino acid in rabbit P450 2E1 with aspartate, arginine, leucine, tryptophan, or tyrosine, and the mutant proteins were expressed in Escherichia coli . Although immunoblot analysis of whole cell lysates demonstrated that all P450 proteins (mutants and wild-type) were equally well expressed on a per cell basis, in solubilized membranes only the tryptophan and tyrosine mutants yielded ferrous-CO difference spectra characteristic of P450 . The specific content (nanomoles per milligram of membrane protein) and yield per liter of the Trp mutant holoenzyme were approximately one-third those of the native enzyme, suggesting that heme incorporation was hindered by tryptophan at this position, whereas the specific content and yield per liter of the Tyr mutant were significantly greater than those of the native preparation . The stability of the Trp and Tyr mutants, as judged by thermal denaturation studies, was not different from that of the native enzyme . The Trp mutant had 38% of the aniline hydroxylase activity, 25% of the p-nitrophenol hydroxylase activity, and 39% of the N-nitrosodimethylamine demethylase activity of the native enzyme, demonstrating that this substitution also decreased catalytic activity.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1994 May 17, 33(19), 5912 - 9 Assignment of disulfide bond location in prothoracicotropic hormone of the silkworm, Bombyx mori: a homodimeric peptide; Ishibashi J et al.; The disulfide bond location of a homodimeric peptide, prothoracicotropic hormone (PTTH) of the silkworm, Bombyx mori, was determined by a combination of partial reduction and sequence analysis of peptide fragments generated through a partial reduction of PTTH followed by alkylation and enzyme digestion . The partial reduction and S-alkylation broke the interchain disulfide bond but did not affect the intrachain disulfide bonds, generating monomeric PTTH whose intrachain disulfide bonds were kept intact . This monomeric PTTH has about one-half the biological activity of intact PTTH . Sequence analysis of the fragments generated by lysyl endopeptidase digestion of this monomeric PTTH after complete reduction and S-alkylation by another S-alkylating reagent showed that only the Cys15 residue was reduced and S-alkylated by the foregoing partial reduction, indicating that this residue formed the interchain disulfide bond . The other disulfide bonds which formed intrachain bridgings were determined by sequence and mass analyses of the fragments generated by two successive enzyme digestions of the monomeric PTTH . In conclusion, the disulfide bond location of PTTH was assigned to Cys15-Cys15' as an interchain disulfide linkage and Cys17-Cys54, Cys40-Cys96, and Cys48-Cys98 as intrachain disulfide linkages. Biochemistry, 1994 May 17, 33(19), 5858 - 66 Kinetic characterization of the chemotactic protein from Escherichia coli, CheY . Kinetic analysis of the inverse hydrophobic effect; Munoz V et al.; CheY, the 129 amino acid chemotactic protein from Escherichia coli, is a good model for studying the folding process of the parallel alpha/beta family of proteins . A study of the folding kinetics of CheY using fluorescence and far-UV circular dichroism (CD) stopped-flow measurements is reported . CheY has three prolines, two of them in the trans conformation and one, Pro110, with a cis Lys-Pro peptide bond . This protein presents a unimolecular, but complex, kinetic mechanism that is dominated by a slow phase compatible with a trans-cis isomerization . Mutation of Pro110 to Gly results in the disappearance of this slow phase, indicating that this cis prolyl bond is responsible for it . The slow phase is catalyzed in a very inefficient way by prolyl isomerase, indicating that the cis bond is poorly accessible to the enzyme during refolding . In agreement with this is the fact that the isomerization of the Lys109-Pro110 bond occurs in an intermediate which contains 96% of the native far-UV CD signal and 80% of the native fluorescence signal . Analysis of the unfolded protein with all its prolines in the native conformation shows the existence of a very stable intermediate in the folding reaction . Mutation of a hyperexposed hydrophobic residue, Phe14, to Asn results in an increase in the free energy of unfolding of the protein of approximately 3 kcal mol-1 . Kinetic analysis of the unfolding and refolding reactions of this mutant indicates that the major stabilization effect comes from the relative destabilization of the unfolded state and the kinetic intermediate with respect to the transition state, providing kinetic evidence for the inverse hydrophobic effect . This could also indicate the existence of nonnative interactions in folding intermediates. Biochemistry, 1994 May 17, 33(19), 5846 - 57 Pentalenene synthase . Purification, molecular cloning, sequencing, and high-level expression in Escherichia coli of a terpenoid cyclase from Streptomyces UC5319; Cane DE et al.; Pentalenene synthase, which catalyzes the cyclization of farnesyl diphosphate (1) to the tricyclic sesquiterpene hydrocarbon pentalenene (2), was purified from Streptomyces UC5319 . A 450-bp hybridization probe, generated by PCR amplification of genomic DNA using primers based on N-terminal and internal tryptic peptide sequence data for pentalenene synthase, was used to screen both plasmid and phage DNA libraries of Streptomyces genomic DNA, resulting in the isolation and sequencing of the complete pentalenene synthase gene . PCR was used to insert the pentalenene synthase gene into the T7 expression vector pLM1 . Cloning of the resulting construct in the expression host Escherichia coli BL21 (DE3) gave transformants that expressed pentalenene synthase as greater than 10% of soluble protein . The recombinant enzyme has been purified, and initial physical and kinetic characterization has been performed . The recombinant enzyme appears to be identical in every respect with the native Streptomyces synthase and exhibits the following steady-state kinetic parameters: Km = 0.31 +/- 0.05 microM, kcat = 0.32 +/- s-1, KI(PPi) = 3.2 +/- 0.6 microM . Both enzymes have an absolute requirement of Mg2+ for catalysis and an optimum pH of 8.2-8.4 . Both proteins have M(r) values of 41-42 kDa, as determined by SDS-PAGE. Biochemistry, 1994 May 17, 33(19), 5813 - 8 Recombinant human CNTF receptor alpha: production, binding stoichiometry, and characterization of its activity as a diffusible factor; Panayotatos N et al.; The primary ligand-binding protein (CNTFR alpha) of the multicomponent receptor for ciliary neurotrophic factor was produced in Escherichia coli . Using novel applications of size-exclusion chromatography and a protein gel-shift assay, we obtained quantitative separation of correctly refolded protein, as well as analytical monitoring of the refolding process and ligand binding . By these and other methods, we determined a 1:1 stoichiometry for the receptor-ligand complex . To investigate the proposed activity and mechanism of soluble CNTFR alpha as a diffusible factor, we studied the response of TF-1 cells which lack CNTFR alpha to various CNTF ligands and the stimulation of this response by sCNTFR alpha . The results show that sCNTFR alpha combines with CNTF and mediates cell survival with the same relative ligand specificity and relative affinity as the cell-surface form . Thus, soluble receptor can reconstitute on a cell surface active complexes that are analogous to the native complexes . Moreover, both the relative ligand potency in the absence of CNTFR alpha and the kinetics of the response to sCNTFR alpha indicate that the other components of the receptor complex contribute little, but measurably, to the specific potency of CNTF. Biochemistry, 1994 May 17, 33(19), 5721 - 7 Structure of the NADPH-binding motif of glutathione reductase: efficiency determined by evolution; Rescigno M et al.; The role of the second glycine residue (Gly-176) of the conserved GXGXXA "fingerprint" motif in the NADPH-binding domain of Escherichia coli glutathione reductase has been studied by means of site-directed mutagenesis . This glycine residue occurs at the N-terminus of the alpha-helix in the beta alpha beta fold that characterizes the dinucleotide-binding domain, in close proximity to the pyrophosphate bridge of the bound coenzyme . Introducing an alanine residue (G176A), the minimum possible change, at this position virtually inactivated the enzyme, as did the introduction of valine, leucine, isoleucine, glutamic acid, histidine, or arginine residues . Only the replacement by serine--a natural substitute for this glycine residue in some forms of mercuric reductase, a related flavoprotein disulfide oxidoreductase--produced a mutant enzyme (G176S) that retained significant catalytic activity . It is conceivable that this is due to a favorable hydrogen bond being formed between the serine hydroxyl and a pyrophosphate oxygen atom . In most of the mutant enzymes, the Km for NADPH was substantially greater than that found for wild-type glutathione reductase, as expected, but this was accompanied by an unexpected decrease in the Km for GSSG . The latter can be explained by the observation that the reduction of the enzyme by NADPH, the first half-reaction of the ping-pong mechanism, had become a rate-limiting step of the overall reaction catalyzed, albeit poorly, by the mutant enzymes.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1994 May 17, 33(19), 5711 - 20 The mutant Escherichia coli F112W cyclophilin binds cyclosporin A in nearly identical conformation as human cyclophilin; Fejzo J et al.; The periplasmic Escherichia coli cyclophilin is distantly related to human cyclophilin (34% sequence identity) . Peptidyl-prolyl isomerase activity, cyclosporin A binding, and inhibition of the calcium-dependent phosphatase calcineurin are compared for human and E . coli wild-type and mutant proteins . Like human cyclophilin, the E . coli protein is a cis-trans peptidyl-prolyl isomerase . However, while the human protein binds cyclosporin A tightly (Kd = 17 nM), the E . coli protein does not (Kd = 3.4 microM) . The mutant F112W E . coli cyclophilin has enhanced cyclosporin binding (Kd = 170 nM) . As for the human protein, the complex of the E . coli mutant with cyclosporin A inhibits calcineurin . Here we describe the structure at pH 6.2 of cyclosporin A bound to the mutant E . coli cyclophilin as solved with solution NMR methods . Despite the low overall sequence identity, the structure of the bound cyclosporin A is virtually identical in both proteins . To assess differences of the cyclosporin binding site, the solution structure of wild-type E . coli cyclophilin was compared with structures of uncomplexed human cyclophilin A and with cyclosporin bound . Despite the structural similarity of bound cyclosporin A, the architecture of the binding site in the E . coli protein is substantially different at the site most distant to tryptophan 121 (human sequence) . This site is constructed by a five-residue insertion in a loop of the E . coli protein, replacing another loop in the human protein. Biochemistry, 1994 May 17, 33(19), 5674 - 81 Kilobase-range communication between polypurine.polypyrimidine tracts in linear plasmids mediated by triplex formation: a braided knot between two linear duplexes; Hampel KJ et al.; Linear plasmids were constructed containing two pyrimidine tracts that were 0.34 and 0.94 kilobases (kb) from either end and were separated by 2.8 kb . The tracts {d(TCCTTC)n and d(CTTCCT)n where n = 6 or 12} were designed so as to be able to form triplexes with each other but not with themselves . Upon lowering of the pH to 4 in the presence of spermine, these plasmids form intermolecular dimers and intramolecular loops of 2.8 kb, as judged from mobility changes on agarose gels . A tethered loop could also be formed in a linear plasmid containing two identical tracts by adding an homologous single-stranded oligopyrimidine, but not an oligopurine . In plasmids containing different tracts, the formation of both dimers and loops could be blocked by adding either homologous single-stranded oligopyrimidine but not an oligopurine . Together with the requirement of low pH, these results demonstrate that triplex formation is of the pyr.pur.pyr type . The extent of dimer and loop formation was dependent on the length of the pyrimidine tract: dimers could be detected in plasmids containing the 72 base pair (bp) inserts after incubation at pH 6, but in plasmids containing the 36 bp inserts, a pH of 5 was required . Hysteresis was also evident to a remarkable extent . Once formed at pH 4, loops and dimers remained stable indefinitely at pH 8, suggesting that the structures become topologically trapped . However, the structures were resolved into the component linear plasmids by incubation with nuclease P1 . This is the first demonstration of a braided or hydrogen-bonded knot between two linear duplexes and may have implications for chromosomal loop formation. Biochemistry, 1994 May 17, 33(19), 5984 - 6003 Backbone dynamics of a free and phosphopeptide-complexed Src homology 2 domain studied by 15N NMR relaxation; Farrow NA et al.; The backbone dynamics of the C-terminal SH2 domain of phospholipase C gamma 1 have been investigated . Two forms of the domain were studied, one in complex with a high-affinity binding peptide derived from the platelet-derived growth factor receptor and the other in the absence of this peptide . 2-D 1H-15N NMR methods, employing pulsed field gradients, were used to determine steady-state 1H-15N NOE values and T1 and T2 15N relaxation times . Backbone dynamics were characterized by the overall correlation time (tau m), order parameters (S2), effective correlation times for internal motions (tau e), and, if required, terms to account for motions on a microsecond-to-millisecond-time scale . An extended two-time-scale formalism was used for residues having relaxation data and that could not be fit adequately using a single-time-scale formalism . The overall correlation times of the uncomplexed and complexed forms of SH2 were found to be 9.2 and 6.5 ns, respectively, suggesting that the uncomplexed form is in a monomer-dimer equilibrium . This was subsequently confirmed by hydrodynamic measurements . Analysis of order parameters reveals that residues in the so-called phosphotyrosine-binding loop exhibited higher than average disorder in both forms of SH2 . Although localized differences in order parameters were observed between the uncomplexed and complexed forms of SH2, overall, higher order parameters were not found in the peptide-bound form, indicating that on average, picosecond-time-scale disorder is not reduced upon binding peptide . The relaxation data of the SH2-phosphopeptide complex were fit with fewer exchange terms than the uncomplexed form . This may reflect the monomer-dimer equilibrium that exists in the uncomplexed form or may indicate that the complexed form has lower conformational flexibility on a microsecond-to-millisecond-time scale. J Immunol Methods, 1994 May 16, 171(2), 157 - 67 Determination of haptoglobin expression in IL-6 treated HepG2 cells by ELISA and by RNA hybridization--evaluation of a quantitative method to measure IL-6; Boe A et al.; Interleukin-6 (IL-6) is known to be an important modulator of acute phase (AP) protein expression in hepatocytes both in vivo and in vitro . In the present study the inducing activity of IL-6 on the expression of the AP protein haptoglobin (HP) by the human hepatoma cell line HepG2, has been evaluated . HP mRNA inducibility was analysed by Northern and slot-blot hybridization, while HP protein was detected by means of an ELISA procedure . A dose-response relationship from 0.3 to 4.8 ng/ml of a human recombinant IL-6 preparation derived from a Chinese hamster ovary (CHO) cell line was observed after 48 h of treatment . Comparable results were obtained by analysing both HP mRNA expression and HP protein secretion . Detectable induction of HP protein secretion was observed with as little as 25 pg/ml of IL-6 . The effect of IL-6 was potentiated by dexamethasone, while an inhibition on HP mRNA inducibility could be prevented by lowering the foetal calf serum (FCS) concentration to 1% . Preliminary data indicate that neither IL-1 beta nor TNF-alpha were able to induce significantly HP mRNA expression and protein secretion . The activity ratio between two IL-6 preparations (from CHO and E . coli cells) obtained with a conventional IL-6 bioassay (i.e., T1165 cell growth assay) was comparable to that obtained in the induction of HP expression . The nominal specific activity of the CHO-derived IL-6 was two to three times higher with both responses. FEBS Lett, 1994 May 16, 344(2-3), 187 - 90 Intergenic suppression in a beta subunit mutant with defective assembly in Escherichia coli F1ATPase . Second-site mutation in the alpha subunit; Miki J et al.; Substitution of Leu-40 by Pro in the beta subunit (beta L40P) of Escherichia coli F1-ATPase caused a decrease in the amount of the alpha and beta subunits on the membranes . A revertant strain, Re50, carrying no suppression mutations in the uncD gene encoding the beta subunit, was isolated from the beta L40P mutant . The uncA gene from this revertant was amplified by PCR, and cloned into an expression plasmid . The expression plasmid carrying the uncA gene from the revertant was used for genetic suppression assays . The suppression mutation in Re50 was in the alpha subunit, and it recovered the assembly of the alpha and beta subunits into the F1F0 complex and the ATPase activity to 50% that of the wild type . In Re50, Leu-111 was substituted by Gln in the alpha subunit . These results suggest that the regions including Leu-40 in the beta subunit and Leu-111 in the alpha subunit are located close together and interact with each other, either directly or indirectly. FEBS Lett, 1994 May 16, 344(2-3), 181 - 6 Characterization of cytosolic malic enzyme in human tumor cells; Loeber G et al.; Cytosolic NADP(+)-dependent malic enzyme (ME) from human tumor cells was characterized in detail and compared to ME from normal human tissues produced in recombinant E . coli . Kinetic properties, size as seen in SDS gels, and HPLC elution profiles of tryptic digests of human 'normal cell' ME and NADP(+)-ME from tumor cells were identical . Thus, NADP(+)-ME found in tumor cells does not constitute a tumor-specific isoform as suggested by other studies but is identical to the 'housekeeping protein' predominantly expressed in human liver and white adipose tissue. FEBS Lett, 1994 May 16, 344(2-3), 171 - 4 Site-specific incorporation of photofunctional nonnatural amino acids into a polypeptide through in vitro protein biosynthesis; Hohsaka T et al.; Nonnatural amino acids with photofunctional groups were incorporated site-specifically into a polypeptide by using in vitro protein synthesizing system . The nonnatural amino acids were attached to tRNA(CCU) through chemical misacylation method, and added to the in vitro system with a mRNA containing a single AGG codon . L-p-Phenylazophenylalanine, L-2-anthrylalanine, L-1-naphthylalanine, L-2-naphthylalanine and L-p-biphenylalanine were successfully incorporated into a polypeptide, but l-1-pyrenylalanine was not . The polypeptides containing the nonnatural amino acids showed photofunctionalities. FEBS Lett, 1994 May 16, 344(2-3), 166 - 70 Nucleic acid-binding regions of the second-largest subunit of Drosophila RNA polymerase II identified by southwestern blotting; Kontermann RE et al.; Analysing overlapping bacterially expressed fragments of the second-largest subunit of Drosophila melanogaster RNA polymerase II in Southwestern DNA binding assays we have identified regions that have the potential to bind nucleic acids non-specifically . A region exhibiting strong DNA binding is located in the N-terminal part of the molecule (amino acids 357-504) and some weak DNA binding is observed for the C-terminal part (amino acids 860-1160) . The non-specific DNA binding behavior of these regions is similar to that of the native enzyme . Most of the known mutations responsible for rifampicin resistance map to a region of the Escherichia coli beta subunit corresponding to the N-terminal nucleic acid-binding region, indirectly supporting the notion that this region participates in interaction with the RNA transcript in ternary complexes. Biochem Biophys Res Commun, 1994 May 16, 200(3), 1477 - 83 Carboxy-terminal region of Escherichia coli SecA ATPase is important to promote its protein translocation activity in vivo; Rajapandi T et al.; The role of the carboxy-terminal region of E . coli SecA ATPase was investigated by using genetic methods to construct a truncated SecA protein missing the last 66 amino acid residues and by systematically substituting serine for each of four cysteine residues present in this protein . Truncation of SecA or alteration of any of the carboxy-terminal cysteine residues resulted in poor growth and a protein secretion defect, indicating that this region of SecA is important in its protein translocation activity . Biochemical analysis of the altered proteins revealed a modest increase in translocation ATPase activity, suggesting that the carboxy-terminal region of SecA may facilitate the coupling of its ATPase activity to cycles of protein translocation. FEBS Lett, 1994 May 16, 344(2-3), 129 - 35 The stability and hydrophobicity of cytosolic and mitochondrial malate dehydrogenases and their relation to chaperonin-assisted folding; Staniforth RA et al.; mMDH and cMDH are structurally homologous enzymes which show very different responses to chaperonins during folding . The hydrophilic and stable cMDH is bound by cpn60 but released by Mg-ATP alone, while the hydrophobic and unstable mMDH requires both Mg-ATP and cpn10 . Citrate equalises the stability of the native state of the two proteins but has no effect on the co-chaperonin requirement, implying that hydrophobicity, and not stability, is the determining factor . The yield and rate of folding of cMDH is unaffected while that of mMDH is markedly increased by the presence of cpn60, cpn10 and Mg-ATP . In 200 mM orthophosphate, chaperonins do not enhance the rate of folding of mMDH, but in low phosphate concentrations chaperonin-assisted folding is 3-4-times faster. J Immunol Methods, 1994 May 16, 171(2), 211 - 26 Recombinant single-chain antibody peptide conjugates expressed in Escherichia coli for the rapid diagnosis of HIV; Lilley GG et al.; Recombinant single chain Fv (scFv) antibody fragments can form the basis of a rapid, whole-blood diagnostic assay . The scFv described in this study is derived from a monoclonal antibody which has a high affinity for glycophorin A, an abundant glycoprotein on the human red blood cell membrane surface . The prototype reagent built around the scFv was designed to detect, in whole blood samples, the presence of antibodies that have arisen through infection with a foreign organism such as human immunodeficiency virus . The scFv was composed of the antibody heavy-chain variable domain (Vh) joined by a 15 residue linker -(GGGGS)3- to the light-chain variable domain (V1) terminated by either a C-terminal octapeptide tail (FLAG) or a 35 amino acid segment from the gp41 surface glycoprotein of HIV-1 . Constructs were cloned into a Escherichia coli expression vector, pHFA, and expressed in a soluble form into culture supernatant . The product retained anti-glycophorin activity which could be detected directly in culture supernatants by ELISA . Furthermore, the scFv-epitope fusion functioned efficiently in the whole blood agglutination assay and was able to distinguish between HIV-1 positive and negative sera. FEBS Lett, 1994 May 16, 344(2-3), 242 - 6 Receptor-binding domain of human alpha 2-macroglobulin . Expression, folding and biochemical characterization of a high-affinity recombinant derivative; Holtet TL et al.; A recombinant version of the receptor binding domain (RBDv) of human alpha 2-macroglobulin (alpha 2M) has been expressed in E . coli and refolded using a novel iterative procedure . RBDv (Val1299-Ala1451) is extended by 15 residues at the N-terminal side of the Lys1313-Glu papain cleavage site in human alpha 2M . RBDv contains the intra-chain bridge Cys1329-Cys1444 and is soluble and monomeric . Competition experiments with 125I-labelled methylamine-treated alpha 2M reveal that RBDv binds to the placental receptor for transformed alpha 2M with a Kd of 8 nM, i.e . the binding affinity of RBDv is of the same order of magnitude as the intrinsic affinity for binding of one domain in transformed alpha 2M to one receptor molecule. Biochem Biophys Res Commun, 1994 May 16, 200(3), 1221 - 9 Overexpression and characterization of a recombinant form of rat calcineurin A; Haddy A et al.; Overexpression of rat recombinant calcineurin A catalytic subunit in E . coli was achieved using a system under control of the T7 promoter . The specific activity of the purified catalytic subunit was suppressed relative to native bovine calcineurin, with the extent of suppression depending upon the choice of substrate . Addition of calcineurin B subunit stimulated phosphatase activity to one third that of native calcineurin . The metal activators Mn2+ and Ni2+ as well as several anion inhibitors affected both native calcineurin and recombinant calcineurin A activity to the same extent . In addition, calcineurin B was required for inhibition by the immunosuppressive complex FK506-FK506-binding protein. Eur J Biochem, 1994 May 15, 222(1), 75 - 81 No intermediate channelling in stepwise hydrolysis of fluorescein di-beta-D-galactoside by beta-galactosidase; Fieldler F et al.; For the hydrolysis of the two glycosidic bonds of fluorescein di-beta-D-galactoside (FDG) by beta-galactosidase from Escherichia coli, small {Hofmann, J . & Sernetz, M . (1983) Anal . Biochem . 131, 180-186} to dramatic {Huang, Z . (1991) Biochemistry 30, 8535-8540} deviations from simple stepwise substrate-intermediate-product kinetics have been reported . Intermediate channelling, a preferred hydrolysis of the intermediate fluorescein mono-beta-D-galactoside (FMG) formed from FDG at the active site and thus in a favourable position for further reaction, has been postulated . As there were reasons to doubt the previous findings and conclusions, the hydrolysis experiments have been repeated at initial FDG concentrations of 7-200 microM, following the concentrations of FDG, FMG and fluorescein with a reliable method, quantitative HPLC, to completion of the reaction . The transient appearance of substantial amounts of the intermediate FMG also in experiments with 200 microM FDG already rules out the existence of the most efficient intermediate channelling deduced by Huang (1991) from measurements of the initially developing fluorescence, incorrectly ascribed to fluorescein . Redetermination of the Michaelis constants for FDG and FMG led to much higher values than those reported previously . Fitting the progress curves by means of nonlinear regression combined with numerical integration of the rate equations resulted in good fits of the normal stepwise substrate-intermediate-product mechanism, without any necessity of assuming a more complex course of the reaction . So one of the rare examples of the hydrolysis of two bonds at a single enzyme-substrate encounter has been invalidated. Eur J Biochem, 1994 May 15, 222(1), 65 - 73 Mapping of Hsp70-binding sites on protein antigens; Roman E et al.; Hsp70-binding sites were mapped on three antigens, the 16-, 19- and 38-kDa proteins of Mycobacterium tuberculosis, using overlapping synthetic peptides in a competitive-binding assay . In each protein, two or three prominent hsp70-binding sites were identified when peptides 20-amino-acid long were used, predominantly in regions containing clusters of aliphatic amino acids . Although there was an overall concordance in the pattern of peptide binding to hsp70 from bacterial (M . tuberculosis) and mammalian sources (immunoglobulin heavy-chain-binding protein), some differences in the specificity of polypeptide binding and the effect of peptides on ATPase activity were observed. Eur J Biochem, 1994 May 15, 222(1), 49 - 54 Assembly of functional skeletal muscle troponin complex in Escherichia coli; Malnic B et al.; The production of multi-subunit proteins of eukaryotic origin in Escherichia coli usually relies on the different subunits being expressed individually and the protein being reassembled in vitro . Here we describe the construction and characterization of plasmids capable of coexpressing the three subunits of chicken skeletal muscle troponin complex in E . coli . We demonstrate that the troponin subunits assembled in the cytoplasm of E . coli cell are fully functional . The troponin complex was purified to homogeneity in high yields . When reconstituted into actin filaments, the complex assembled in vivo was capable of regulating the myosin ATPase with a calcium dependence that was identical to the complex reconstituted in vitro . These results demonstrate that the coexpression of the subunits of a protein complex can prevent the accumulation of denatured proteins in inclusion granules. Eur J Biochem, 1994 May 15, 222(1), 27 - 32 The NAM1 protein (NAM1p), which is selectively required for cox1, cytb and atp6 transcript processing/stabilisation, is located in the yeast mitochondrial matrix; Wallis MG et al.; The NAM1 nuclear gene was shown to control the stability and/or processing of mitochondrial transcripts of the cytochrome b, cytochrome oxidase subunit I and ATP synthase subunit VI genes {Groudinsky O., Bousquet I., Wallis M . G., Slonimski, P . P . & Dujardin G . (1993) Mol . Gen . Genet . 240, 419-427} . In order to better understand the mode of action of the NAM1 gene product, we have examined its intracellular fate . A fusion plasmid enabling bacterial over-expression of the corresponding protein-A-NAM1 cognate was constructed and subsequently employed as an antigen to raise polyclonal antibodies . These antibodies specifically recognise a 50-kDa protein which purifies along with the mitochondria and corresponds to NAM1p . Submitochondrial localisation experiments show that NAM1p is a soluble protein, located interior to the mitoplasts . Matricial location is a strong argument in favour of a direct interaction of NAM1p with particular mitochondrial transcripts and leads us to propose a model in which NAM1p could be an RNA-convoying protein stabilising and directing mitochondrial transcripts towards the inner face of the inner membrane where translation and assembly seem to occur. Eur J Biochem, 1994 May 15, 222(1), 173 - 81 Solid-phase synthesis, conformational analysis and in vitro cleavage of synthetic human synaptobrevin II 1-93 by tetanus toxin L chain; Cornille F et al.; A 93-residue peptide corresponding to the cytosolic domain of a human vesicle associated membrane protein (VAMP or synaptobrevin) has been prepared by solid-phase peptide synthesis in order to investigate the proteolytic activity of the tetanus toxin light chain (TeTx L chain) . This protein has been recently reported to inactivate the neuronal rat synaptobrevin II by proteolysis . We show in this study that the synthetic human synaptobrevin II 1-93 (Syb II 1-93) as well as an N-terminus-shortened 69-residue peptide (Syb II 25-93) were cleaved selectively at the Gln76-Phe77 peptide bond by TeTx L chain while shorter peptides were not . A Michaelis constant Km = 192 +/- 2 microM and a catalytic constant kcat = 0.5 min-1 were found for the 93-residue peptide . A neutral optimum pH for the cleavage rate, an inhibition by preincubation of the toxin with well known nonspecific inhibitors of metallopeptidases as well as a zinc-dependent enzyme activity suggest that TeTx belongs to the zinc endopeptidase family . Moreover an activation by reducing agents and an inhibition by cysteine-modifying chemical reagents indicate a critical thiol dependency . Among several specific inhibitors of zinc endopeptidases tested, none could inhibit TeTx L chain even at high concentration . Structural studies by 600-MHz 1H-NMR showed that in water or dimethylsulfoxide the peptide Syb II 1-93 and shorter fragments did not present well defined conformations . Nevertheless protein-protein interactions have been shown for the peptides Syb II 1-93 and 25-93 but not for Syb II 51-93, a fragment not cleaved by TeTx L chain. Eur J Biochem, 1994 May 15, 222(1), 137 - 46 Characterization of a polyhistidine-tagged form of human myristoyl-CoA: protein N-myristoyltransferase produced in Escherichia coli; McIlhinney RA et al.; The enzyme myristoyl-CoA:protein N-myristoyltransferase is responsible for the attachment of a myristoyl group to the N-terminal glycine of a number of cell, viral and fungal proteins . In order to overcome the difficulties of purification of this enzyme from tissue sources, we have produced an N-terminally polyhistidine-tagged version of the enzyme and expressed this in Escherichia coli . The resulting enzyme has a molecular mass of 53 kDa and is fully active showing the expected specificity for myristic acid and causing the N-terminal myristoylation of both synthetic peptide and protein substrates in vitro . The enzyme exhibits a broad pH optimum peaking at a pH of 8.0 and has a Km for myristoyl-CoA of 7.6 microM . The two synthetic peptide substrates based on the N-terminal sequence of the catalytic subunit of protein kinase A (GNAAAARR) and of p60src (GSSKSKPKDPSQRRRY) have different kinetic parameters with Km values of 115.2 microM and 44.2 microM and Vmax values of 95 and 120 nmol.min-1.mg-1, respectively . The expressed enzyme is partially inhibited (50%) by iodoacetamide at 5 mM and fully inhibited by diethylpyrocarbonate at 10 mM . This latter inhibition can be prevented by including histidine in the incubation of the enzyme and inhibitor . Antisera raised to synthetic peptides based on sequences derived from the N- and C- terminus of the human enzyme reacted with the expressed protein on Western blots, but only the N-terminal sequence reacted with the native protein suggesting that the C-terminus may be not be accessible . The enzyme can catalyse the removal of a myristoyl group from myristoylated peptides but does so only in the presence of added coenzyme A. Biochem J, 1994 May 15, 300 ( Pt 1), 85 - 90 Cloning and expression in Escherichia coli of a dog thyroid cDNA encoding a novel inositol 1,4,5-trisphosphate 5-phosphatase; Verjans B et al.; In brain and many other tissues, type I inositol 1,4,5-trisphosphate (InsP3) 5-phosphatase is the major isoenzyme hydrolysing the calcium-mobilizing second messenger InsP3 . This protein has been purified to apparent homogeneity from a crude soluble fraction of bovine brain, yielding a single major protein band with a molecular mass of 43 kDa after SDS/PAGE . This material was used to determine internal microsequences . A partial DNA sequence has been amplified by PCR by using degenerate primers deduced from two protein sequences (FKAKKYKKV and DENYKSQE) . A cDNA clone (BVCT) was isolated by screening a dog thyroid cDNA library . The encoded protein of 412 amino acids has a calculated molecular mass of 47,681 Da . Peptide sequences generated from the bovine brain enzyme were found to be 96% conserved compared with the dog thyroid protein . When clone BVCT was expressed in Escherichia coli, the recombinant protein was shown to hydrolyse both InsP3 and inositol 1,3,4,5-tetrakisphosphate, with apparent Km values of 28 and 3 microM respectively . Enzyme activity was inhibited by EDTA and 2,3-bisphosphoglycerate, both inhibitors of native InsP3 5-phosphatase, but not by EGTA and LiCl, as previously shown for the bovine brain enzyme . Our data show the cloning of type I InsP3 5-phosphatase which, interestingly, does not share any significant sequence identity with the previously cloned type III isoenzyme. Biochem J, 1994 May 15, 300 ( Pt 1), 111 - 5 Site-directed mutagenesis of rat muscle 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: role of Asp-130 in the 2-kinase domain; Rider MH et al.; Asp-130 of the recombinant skeletal-muscle 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase was mutated into Ala in order to study its role in catalysis and/or substrate binding . The D130A mutant displayed a 30- to 140-fold decreased 2-kinase Vmax, depending on the pH, and a 30- and 60-fold increase in Km for MgATP and Fru-6-P respectively at pH 8.5 compared with the wild-type . Mutagenesis of Asp-130 to Ala had no effect on the 2-phosphatase activity, and fluorescence measurements indicated that the changes in kinetic properties of PFK-2 in the D130A mutant were not due to instability . The role of Asp-130 in the 2-kinase reaction is discussed and compared with that of Asp-103 of 6-phosphofructo-1-kinase from Escherichia coli, which binds Mg2+. EMBO J, 1994 May 15, 13(10), 2464 - 71 Many of the conserved nucleotides of tRNA(Phe) are not essential for ternary complex formation and peptide elongation; Nazarenko IA et al.; An RNase protection assay was used to show that the dissociation rate constants and equilibrium constants of unmodified yeast and Escherichia coli phenylalanyl-tRNA(Phes) to elongation factor Tu from E.coli were very similar to each other and to their fully modified counterparts . The affinity of aminoacylated tRNA to elongation factor Tu was substantially lower when GTP analogues were used in place of GTP, emphasizing the importance of the beta-gamma phosphate linkage in the function of G-proteins . Fourteen different mutations in conserved and semi-conserved nucleotides of yeast phenylalanyl-tRNA(Phe) were tested for binding to elongation factor Tu.GTP and assayed for activity in the ribosomal A- and P-sites . Most of the mutations did not severely impair the function of these tRNAs in any of the assays . This suggests that the translational machinery does not form sequence-specific interactions with the conserved nucleotides of tRNA. EMBO J, 1994 May 15, 13(10), 2421 - 31 Functional association of essential splicing factor(s) with PRP19 in a protein complex; Tarn WY et al.; We have previously shown that the yeast PRP19 protein is a spliceosomal component, but is not tightly associated with small nuclear RNAs . It appears to associate with the spliceosome concomitant with or just after dissociation of the U4 small nuclear RNA during spliceosome assembly . We have found that PRP19 is associated with a protein complex in the splicing extract and that at least one of the associated components is essential for splicing . Taking advantage of the epitope tagging technique, we have isolated the PRP19-associated complex by affinity chromatography . The isolated complex is functional for complementation for the heat-inactivated prp19 mutant extract, and consists of at least seven polypeptides in addition to PRP19 . At least three of these can interact directly with the PRP19 protein . We also show that the PRP19 protein itself is in an oligomeric form, which might be a prerequisite for its interaction with these proteins. EMBO J, 1994 May 15, 13(10), 2267 - 72 Membrane protein topology: effects of delta mu H+ on the translocation of charged residues explain the 'positive inside' rule; Andersson H et al.; The membrane electrochemical potential is critical for the export of most periplasmic proteins in Escherichia coli . Its exact role during insertion of integral inner membrane proteins, however, remains obscure . Using derivatives of the inner m