Biomedical subjects

M Grunberg-Manago

Publications and source records attributed to M Grunberg-Manago.

At least 37 records · Page 2Linked to original sources

PubMed1994

Mutational analysis of the pseudoknot structure of the S15 translational operator from Escherichia coli.

Expression of rpsO, the gene encoding the small ribosomal protein S15, is autoregulated at the translational level by S15, which binds to its mRNA in a region overlapping the ribosome-binding site. By measuring the effect of mutations on the expression of a translational rpsO-lacZ fusion and the S15 binding affinity for the translational operator, the formation of a pseudoknot in the operator site in vivo is fully demonstrated and appears to be a prerequisite for S15 binding. The mutational analysis suggests also that specific determinants for S15 binding are located in very limited regions of the structure formed by the pseudoknot. It is deduced that a specific pseudoknot conformation is a key element for autoregulation.

Bacteriophage lambda↗

PubMed1994

Expression of both Bacillus subtilis threonyl-tRNA synthetase genes is autogenously regulated.

The "housekeeping" threonyl-tRNA synthetase gene (thrS) of Bacillus subtilis is shown to be transcribed in vivo and in vitro from a single promoter. In vitro, 85% of all messages transcribed from the thrS promoter are terminated at a strong factor-independent terminator localized upstream of the thrS Shine-Dalgarno sequence, within the 305-nucleotide-long leader region. Overexpression of thrS represses transcriptional and translational thrS-lacZ fusions to a similar extent, suggesting that thrS is autoregulated at the transcriptional level. We show that autogenous control does not act at the level of transcription initiation but involves antitermination of the transcription mechanism. thrZ, the second threonyl-tRNA synthetase gene, is also autogenously regulated. However, the ability of the ThrS synthetase to repress thrS as well as thrZ expression is much greater than that of the ThrZ synthetase.

Bacillus subtilis↗

PubMed1993

Translational regulation of the Escherichia coli threonyl-tRNA synthetase gene: structural and functional importance of the thrS operator domains.

Previous work showed that E coli threonyl-tRNA synthetase (ThrRS) binds to the leader region of its own mRNA and represses its translation by blocking ribosome binding. The operator consists of four distinct domains, one of them (domain 2) sharing structural analogies with the anticodon arm of the E coli tRNA(Thr). The regulation specificity can be switched by using tRNA identity rules, suggesting that the operator could be recognized by ThrRS as a tRNA-like structure. In the present paper, we investigated the relative contribution of the four domains to the regulation process by using deletions and point mutations. This was achieved by testing the effects of the mutations on RNA conformation (by probing experiments), on ThrRS recognition (by footprinting experiments and measure of the competition with tRNA(Thr) for aminoacylation), on ribosome binding and ribosome/ThrRS competition (by toeprinting experiments). It turns out that: i) the four domains are structurally and functionally independent; ii) domain 2 is essential for regulation and contains the major structural determinants for ThrRS binding; iii) domain 4 is involved in control and ThrRS recognition, but to a lesser degree than domain 2. However, the previously described analogies with the acceptor-like stem are not functionally significant. How it is recognized by ThrRS remains to be resolved; iv) domain 1, which contains the ribosome loading site, is not involved in ThrRS recognition. The binding of ThrRS probably masks the ribosome binding site by steric hindrance and not by direct contacts. This is only achieved when ThrRS interacts with both domains 2 and 4; and v) the unpaired domain 3, which connects domains 2 and 4, is not directly involved in ThrRS recognition. It should serve as an articulation to provide an appropriate spacing between domains 2 and 4. Furthermore, it is possibly involved in ribosome binding.

Base Sequence↗

PubMed1993

Similar organization of the nusA-infB operon in Bacillus subtilis and Escherichia coli.

We reported previously the cloning and sequence of the Bacillus subtilis infB gene which encodes the essential IF2 factor required for initiation of translation (K. Shazand, J. Tucker, R. Chiang, K. Stansmore, H. U. Sperling-Petersen, M. Grunberg-Manago, J. C. Rabinowitz, and T. Leighton, J. Bacteriol. 172:2675-2687, 1990). The location of the 5' border of the infB operon was investigated by using integrative plasmids carrying various DNA fragments from the region upstream of the infB gene. The lethal effect of disruption of the infB transcriptional unit could be suppressed when the integrated plasmid introduced the spac promoter upstream of the infB operon and transformants were selected in conditions of induction of spac expression. Such an integrated plasmid was used as a starting point to clone the promoter of the infB operon. Primer extension mapping suggests that a single sigma A-type promoter controls transcription of the infB operon. The sequence of a 5,760-bp region encompassing the infB gene was determined. The infB operon is located immediately downstream of the polC gene and comprises seven open reading frames, four of which appear to be the homologs of genes present in the same order in the Escherichia coli infB operon, including nusA. The striking similarity between the E. coli and B. subtilis infB operons suggests that the function of each gene pair is conserved and that the B. subtilis NusA homolog, which is 124 residues shorter than its E. coli counterpart, could play a role similar to its role in E. coli.

Amino Acid Sequence↗

PubMed1992

Molecular mimicry in translational control of E. coli threonyl-tRNA synthetase gene. Competitive inhibition in tRNA aminoacylation and operator-repressor recognition switch using tRNA identity rules.

We previously showed that: (i) E.coli threonyl-tRNA synthetase (ThrRS) binds to the leader of its mRNA and represses translation by preventing ribosome binding to its loading site; (ii) the translational operator shares sequence and structure similarities with tRNA(Thr); (iii) it is possible to switch the specificity of the translational control from ThrRS to methionyl-tRNA synthetase (MetRS) by changing the CGU anticodon-like sequence to CAU, the tRNA(Met) anticodon. Here, we show that the wild type (CGU) and the mutated (CAU) operators act as competitive inhibitors of tRNA(Thr) and tRNA(fMet) for aminoacylation catalyzed by E.coli ThrRS and MetRS, respectively. The apparent Kd of the MetRS/CAU operator complex is one order magnitude higher than that of the ThrRS/CGU operator complex. Although ThrRS and MetRS shield the anticodon- and acceptor-like domains of their respective operators, the relative contribution of these two domains differs significantly. As in the threonine system, the interaction of MetRS with the CAU operator occludes ribosome binding to its loading site. The present data demonstrate that the anticodon-like sequence is one major determinant for the identity of the operator and the regulation specificity. It further shows that the tRNA-like operator obeys to tRNA identity rules.

Acylation↗

PubMed1992

Tandem translation of Bacillus subtilis initiation factor IF2 in E. coli. Over-expression of infBB.su in E. coli and purification of alpha- and beta-forms of IF2B.su.

The protein synthesis initiation factor, IF2, in Bacillus subtilis has previously been characterized as being present in two forms, alpha and beta, of molecular mass 79 and 68 kDa, respectively, on the basis of their cross-reaction with anti-E. coli IF2 antibodies and by the DNA sequence of the gene for IF2, infBB.su. In this work we have cloned infBB.su in E. coli cells. Two proteins of molecular mass identical to the B. subtilis IF2 alpha and -beta were over-expressed and purified using a new three-step ion-exchange chromatography procedure. The N-terminal amino acid sequence of the two proteins was determined and the results confirmed that the two forms were IF2 alpha and -beta, both encoded by the infB gene. The N-terminal amino acid sequence determined for IF2 beta is Met94-Gln-Asn-Asn-Gln-Phe. The presence of methionine at position 94 shows that this form is, in fact, the result of a second translational initiation in infBB.su mRNA, since the codon at amino acid position 94 is GUG, which is the normal codon for valine, but also known to be an initiator codon. This is a new example of the unusual tandem translation in E. coli of an open mRNA reading frame.

Amino Acid Sequence↗

PubMed1992

Domains of the Escherichia coli threonyl-tRNA synthetase translational operator and their relation to threonine tRNA isoacceptors.

The expression of the gene for threonyl-tRNA synthetase (thrS) is negatively autoregulated at the translational level in Escherichia coli. The synthetase binds to a region of the thrS leader mRNA upstream from the ribosomal binding site inhibiting subsequent translation. The leader mRNA consists of four structural domains. The present work shows that mutations in these four domains affect expression and/or regulation in different ways. Domain 1, the 3' end of the leader, contains the ribosomal binding site, which appears not to be essential for synthetase binding. Mutations in this domain probably affect regulation by changing the competition between the ribosome and the synthetase for binding to the leader. Domain 2, 3' from the ribosomal binding site, is a stem and loop with structural similarities to the tRNA(Thr) anticodon arm. In tRNAs the anticodon loop is seven nucleotides long, mutations that increase or decrease the length of the anticodon-like loop of domain 2 from seven nucleotides abolish control. The nucleotides in the second and third positions of the anticodon-like sequence are essential for recognition and the nucleotide in the wobble position is not, again like tRNA(Thr). The effect of mutations in domain 3 indicate that it acts as an articulation between domains 2 and 4. Domain 4 is a stable arm that has similarities to the acceptor arm of tRNA(Thr) and is shown to be necessary for regulation. Based on this mutational analysis and previous footprinting experiments, it appears that domains 2 and 4, those analogous to tRNA(Thr), are involved in binding the synthetase which inhibits translation probably by interfering with ribosome loading at the nearby translation initiation site.

Base Sequence↗

PubMed1992

Both forms of translational initiation factor IF2 (alpha and beta) are required for maximal growth of Escherichia coli. Evidence for two translational initiation codons for IF2 beta.

The gene infB codes for two forms of translational initiation factor IF2; IF2 alpha (97,300 Da) and IF2 beta (79,700 Da). IF2 beta arises from an independent translational event on a GUG codon located 471 bases downstream from IF2 alpha start codon. By site-directed mutagenesis we constructed six different mutations of this GUG codon. In all cases, IF2 beta synthesis was variably affected by the mutations but not abolished. We show that the residual expression of IF2 beta results from translational initiation on an AUG codon located 21 bases downstream from the mutated GUG. Furthermore, two forms of IF2 beta have been separated by fast protein liquid chromatography and the determination of their N-terminal sequences indicated that they resulted from two internal initiation events, one occurring on the previously identified GUG start codon, the other on the AUG codon immediately downstream. We conclude that two forms of IF2 beta exist in the cell, which differ by seven aminoacid residues at their N terminus. Only by mutating both IF2 beta start codons could we construct plasmids that express only IF2 alpha. A plasmid expressing only IF2 beta was obtained by deletion of the proximal region of the infB gene. Using a strain that carries a null mutation in the chromosomal copy of infB and a functional copy of the same gene on a thermosensitive lysogenic lambda phage, we could cure the lambda phage when the plasmids expressing only one form of IF2 were supplied in trans. We found that each one of the two forms of IF2, at near physiological levels, can support growth of Escherichia coli, but that growth is retarded at 37 degrees C. This result shows that both forms of IF2 are required for maximal growth of the cell and suggests that they have acquired some specialized but not essential function.

Amino Acid Sequence↗

PubMed1992

Co-ordinate expression of the two threonyl-tRNA synthetase genes in Bacillus subtilis: control by transcriptional antitermination involving a conserved regulatory sequence.

In Bacillus subtilis, two genes, thrS and thrZ, encode distinct threonyl-tRNA synthetase enzymes. Normally, only the thrS gene is expressed. Here we show that either gene, thrS or thrZ, is sufficient for normal cell growth and sporulation. Reducing the intracellular ThrS protein concentration induces thrZ expression in a dose-compensatory manner. Starvation for threonine simultaneously induces thrZ and stimulates thrS expression. The 5'-leader sequences of thrS and thrZ contain, respectively, one and three transcription terminators preceded by a conserved sequence. We show that this sequence is essential for the regulation of thrS via a transcriptional antitermination mechanism. We propose that both genes, thrS and thrZ, are regulated by the same mechanism such that the additional regulatory domains present before thrZ account for its non-expression. In contrast to Escherichia coli, structurally similar regulatory domains, i.e. the consensus sequence preceding a terminator structure, are found in the leader regions of most aminoacyl-tRNA synthetase genes of Gram-positive bacteria. This suggests that they are regulated by a common mechanism.

Bacillus subtilis↗

PubMed1991

A severely truncated form of translational initiation factor 2 supports growth of Escherichia coli.

We have constructed strains carrying null mutations in the chromosomal copy of the gene for translational initiation factor (IF) 2 (infB). A functional copy of the infB gene is supplied in trans by a thermosensitive lysogenic lambda phage integrated at att lambda. These strains enabled us to test in vivo the importance of different structural elements of IF2 expressed from genetically engineered plasmid constructs. We found that, as expected, the gene for IF2 is essential. However, a protein consisting of the C-terminal 55,000 Mr fragment of the wild-type IF2 protein is sufficient to allow growth when supplied in excess. This result suggests that the catalytic properties are localized in the C-terminal half of the protein, which includes the G-domain, and that this fragment is sufficient to complement the IF2 deficiency in the infB deletion strain.

Bacterial Proteins↗

PubMed1991

Structural and functional domains of E coli initiation factor IF2.

Initiation of translation in prokaryotes requires the participation of at least three soluble proteins: the initiation factors IF1, IF2 and IF3. Initiation factor 2, which is one of the largest proteins involved in translation (97.3 kDa) has been shown to stimulate in vitro the binding of fMet-tRNA(fMet) to the 30S ribosomal subunit. After formation of 70S translation initiation complex, IF2 is believed to participate in GTP hydrolysis, thereby promoting its own release. Here we review evidence which indicates the functional importance of the different structural domains of IF2, emphasizing new information obtained by in vivo experiments.

Amino Acid Sequence↗

PubMed1990

Escherichia coli threonyl-tRNA synthetase and tRNA(Thr) modulate the binding of the ribosome to the translational initiation site of the thrS mRNA.

Escherichia coli threonyl-tRNA synthetase binds to the leader region of its own mRNA at two major sites: the first shares some analogy with the anticodon arm of several tRNA(Thr) isoacceptors and the second corresponds to a stable stem-loop structure upstream from the first one. The binding of the enzyme to its mRNA target site represses its translation by preventing the ribosome from binding to its attachment site. The enzyme is still able to bind to derepressed mRNA mutants resulting from single substitutions in the anticodon-like arm. This binding is restricted to the stem-loop structure of the second site. However, the interaction of the enzyme with this site fails to occlude ribosome binding. tRNA(Thr) is able to displace the wild-type mRNA from the enzyme at both sites and suppresses the inhibitory effect of the synthetase on the formation of the translational initiation complex. Our results show that tRNA(Thr) acts as an antirepressor on the synthesis of its cognate aminoacyl-tRNA synthetase. This repression/derepression double control allows precise adjustment of the rate of synthesis of threonyl-tRNA synthetase to the tRNA level in the cell.

Anticodon↗

PubMed1990

Purified internal G-domain of translational initiation factor IF-2 displays guanine nucleotide binding properties.

Translational initiation factor IF-2 is involved in a multistep pathway leading to the synthesis of the first peptide bond. IF-2 is a guanine nucleotide binding protein (G-protein) and catalyzes GTP hydrolysis in the presence of ribosomes. According to sequence homologies with other G-proteins, particularly EF-Tu, a theoretical model for the tertiary structure of the putative G-domain of IF-2 has been previously proposed [Cenatiempo, Y., Deville, F., Dondon, J., Grunberg-Manago, M., Hershey, J. W. B., Hansen, H. F., Petersen, H. U., Clark, B. F. C., Kjeldgaard, M., La Cour, T. F. M., Mortensen, K. K., & Nyborg, J. (1987) Biochemistry 26, 5070-5076]. A short fragment of IF-2 encompassing the putative G-domain was purified by limited proteolysis of a chimeric protein, synthesized from a gene fusion, between a segment of the IF-2 gene and lacZ. The N- and C-terminal sequences of this IF-2 peptide were characterized. Its calculated length is 181 amino acids and its molecular mass 19.4 kDa, whereas it migrates at 14 kDa in SDS-polyacrylamide gels. This segment of IF-2 can form binary complexes with GDP and can be cross-linked to GTP, therefore indicating that it really corresponds to the G-domain. However, in contrast to the situation described for the purified G-domain of EF-Tu, the IF-2 fragment did not hydrolyze GTP even in the presence of ribosomes. It is assumed that active centers of IF-2 located outside the G-domain are needed for the latter reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1990

The conformation of the initiator tRNA and of the 16S rRNA from Escherichia coli during the formation of the 30S initiation complex.

The conformation of the E. coli initiator tRNA and of the 16S rRNA at different steps leading to the 30S.IF2.fMet-ARN(fMet).AUG.GTP complex has been investigated using several structure-specific probes. As compared to elongator tRNA, the initiator tRNA exhibits specific structural features in the anticodon arm, the T and D loops and the acceptor arm. Initiation factor 2 (IF2) interacts with the T-loop and the minor groove of the T stem of the RNA, and induces an increased flexibility in the anticodon arm. In the 30S initiation complex, additional protection is observed in the acceptor stem and in the anticodon arm of the tRNA. Within the 30S subunit, IF2 does not significantly shield defined portions of 16S rRNA, but induces both reduction and enhancement of reactivity scattered in the entire molecule. Most are constrained in a region corresponding to the cleft, the lateral protrusion and the part of the head facing the protrusion. All the reactivity changes induced by the binding of IF2 are still observed in the presence of the initiator tRNA and AUG message. The additional changes induced by the tRNA are mostly centered around the cleft-head-lateral protrusion region, near positions affected by IF2 binding.

Base Sequence↗

PubMed1990

Translational control of ribosomal protein S15.

The expression of ribosomal protein S15 is shown to be translationally and negatively autocontrolled using a fusion within a reporter gene. Isolation and characterization of several deregulated mutants indicate that the regulatory site (the translational operator site) overlaps the ribosome loading site of the S15 messenger. In this region, three domains, each exhibiting a stem-loop structure, were determined using chemical and enzymatic probes. The most downstream hairpin carries the Shine-Dalgarno sequence and the initiation codon. Genetic and structural data derived from mutants constructed by site-directed mutagenesis show that the operator is a dynamic structure, two domains of which can form a pseudoknot. Binding of S15 to these two domains suggests that the pseudoknot could be stabilized by S15. A model is presented in which two alternative structures would explain the molecular basis of the S15 autocontrol.

Base Sequence↗

PubMed1990

The translational regulation of threonyl-tRNA synthetase. Functional relationship between the enzyme, the cognate tRNA and the ribosome.

The E. coli threonyl-tRNA synthetase gene is negatively autoregulated at the translational level by a direct binding of the enzyme to the leader region of the thrS mRNA. This region folds in four well-defined domains. The enzyme binds to the leader at two major sites: the first is a stem-loop structure located in domain II upstream of the translational initiation site (domain I) which shares structural analogies with the anticodon arm of several tRNA(Thr) isoacceptors. The second site corresponds to a stable stem-loop structure located in domain IV. Both sites are separated by a large unpaired region (domain III). In vivo and in vitro experiments show that the structural integrity of both sites is required for the regulatory process. The binding of the enzyme to its mRNA target site represses its translation by preventing the ribosome from binding to its attachment site. tRNA(Thr) suppresses this inhibitory effect by displacing the mRNA from the enzyme at both the upstream stem-loop structure and the tRNA-like anticodon arm.

Anticodon↗

PubMed1990

Characterization of the translational start site for IF2 beta, a short form of Escherichia coli initiation factor IF2.

The gene for initiation factor IF2, infB, represents one of the few examples in Escherichia coli of genes encoding two protein products in vivo. In a previous work, our group showed that both forms of IF2 (alpha and beta) are closely related and may arise from two independent translational events on infB mRNA. Unambiguous mapping and rigorous determination of the nature of the initiation triplet for IF2 beta, the smaller form of IF2, is critical for future mutagenesis of this codon, required for investigating the biological importance of both IF2 alpha and IF2 beta. Three types of experiments were carried out. First, a 77-bp deletion was created at the beginning of the structural gene leading to premature termination of IF2 alpha synthesis. Under these conditions, IF2 beta is still formed. Second, various Bal31 digests of infB containing the 77-bp deletion were fused to lacZ. Any synthesis of a fused protein with beta-galactosidase activity should reflect the occurrence of an initiation event on the messenger corresponding to this DNA segment. It was consequently possible to locate the IF2 beta initiation site within an 18-base region containing an in-phase GUG codon. Third, to avoid any artefactual reinitiation event possibly occurring under our experimental conditions, we fused to lacZ an infB fragment devoid of IF2 alpha start sequences but containing genetic information for this 18-base region. A hybrid protein with beta-galactosidase activity was synthesized. Moreover, its NH2-terminal amino acid sequence coincided with that of IF2 beta, demonstrating that GUG, located 471 bases downstream from the IF2 alpha external start codon, is the internal start codon for the shorter form of IF2.

Base Sequence↗

PubMed1990

Translational autocontrol of the Escherichia coli ribosomal protein S15.

When rpsO, the gene encoding the ribosomal protein S15 in Escherichia coli, is carried by a multicopy plasmid, the mRNA synthesis rate of S15 increases with the gene dosage but the rate of synthesis of S15 does not rise. A translational fusion between S15 and beta-galactosidase was introduced on the chromosome in a delta lac strain and the expression of beta-galactosidase studied under different conditions. The presence of S15 in trans represses the beta-galactosidase level five- to sixfold, while the synthesis rate of the S15-beta-galactosidase mRNA decreases by only 30 to 50%. These data indicate that S15 is subject to autogenous translational control. Derepressed mutants were isolated and sequenced. All the point mutations map in the second codon of S15, suggesting a location for the operator site that is very near to the translation initiation codon. However, the creation of deletion mutations shows that the operator extends into the 5' non-coding part of the message, thus overlapping the ribosome loading site.

Base Composition↗