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M Grunberg-Manago

Publications and source records attributed to M Grunberg-Manago.

At least 73 records · Page 4Linked to original sources
PubMed1987

Cloning and mapping of infA, the gene for protein synthesis initiation factor IF1.

The gene for translation initiation factor IF1, infA, has been identified by using two synthetic oligonucleotides to screen a Charon 30 library of Escherichia coli DNA. A recombinant lambda phage, C1921, was purified from a plaque positive for both probes. A 2.8 kb BglII fragment and a 2.0 kb HindIII fragment isolated from C1921 were subcloned into the BamHI and HindIII sites of pBR322 to yield pTB7 and pTH2 respectively. Synthesis of IF1 in maxicells transformed with pTB7 or pTH2 indicates the presence of inf A in both inserts. This was confirmed by DNA sequencing: a region was found that codes for a 8,119 dalton protein with an amino acid sequence corresponding to IF1. The chromosomal location of inf A was determined by mapping the closely linked beta-lactamase gene (Ampr) in pTB7 and pTH2. pTB7 and pTH2 were transformed into polA Hfr hosts, and integration of the plasmid by homologous recombination near inf A was selected on the basis of ampicillin resistance. The site of integration was confirmed by Southern blot analysis of restriction nuclease digested wild type and transformed genomic DNA. The Ampr marker (and therefore inf A) was mapped to about 20 minutes by Hfr interrupted matings and P1 transduction experiments. The structure and regulation of the inf A operon currently are being investigated.

Amino Acid Sequence↗
PubMed1987

Nucleotide sequence of the pnp gene of Escherichia coli encoding polynucleotide phosphorylase. Homology of the primary structure of the protein with the RNA-binding domain of ribosomal protein S1.

The pnp gene is located at 69 min on the Escherichia coli chromosome adjacent to the rpsO gene which encodes the ribosomal protein S15. In this paper, we present the sequence of a 3030-nucleotide DNA fragment containing the open reading frames coding for ribosomal protein S15 and polynucleotide phosphorylase. Translation of pnp is initiated by 5'-UUG-3' codon separated by 7 nucleotides from a good ribosome binding site. Codon usage in this gene is typical of highly expressed proteins of E. coli. Some of the transcripts of the pnp gene terminate just after the stem of the terminator t2 visible in the nucleotide sequence. However, a very strong read-through occurs at this site, thus permitting many of the pnp transcripts to extend beyond this transcription terminator. We also describe the primary structure homologies between a 69-amino-acid stretch of polynucleotide phosphorylase and the four homologous stretches of ribosomal protein S1 which form its RNA binding site. The possibility that this 69-amino-acid stretch constitutes the polynucleotide binding domain of polynucleotide phosphorylase is discussed.

Amino Acid Sequence↗
PubMed1987

The first step in the functional inactivation of the Escherichia coli polynucleotide phosphorylase messenger is a ribonuclease III processing at the 5' end.

The transcripts covering pnp, the gene encoding polynucleotide phosphorylase, are processed by ribonuclease III. In this study, it is shown that the steady state level of the pnp mRNA increased 11-fold in a ribonuclease III-deficient strain. The synthesis rate of this messenger is only slightly affected in the mutant strain whereas the half-life, which is 1.5 min in the wild type, is considerably increased to more than 40 min. Moreover, polynucleotide phosphorylase is 10-fold over-expressed in the mutant strain, which shows that unprocessed pnp mRNA is functional. The position of the ribonuclease III-sensitive site suggests that the sequence involved in the stabilization of the pnp mRNA is located at the 5' end of the message and that the RNase III processing triggers the decay of the transcripts downstream. A similar function for ribonuclease III in the processing of the messenger for the beta beta' subunits of RNA polymerase is proposed.

Endoribonucleases↗
PubMed1987

Open reading frames in the control regions of the phenylalanyl-tRNA synthetase operon of E. coli.

The pheST operon codes for the two subunits of phenylalanyl-tRNA synthetase and it expression is controlled by attenuation in a way similar to many amino acid biosynthetic operons. The nucleotide sequence of the control regions of the operon indicates the presence of several open reading frames besides that of the leader peptide. One of these open reading frames, called the alternative leader peptide, starts at about the same place as the leader peptide and ends after the terminator of the attenuator. Another open reading frame, called the terminator peptide, starts after the terminator and covers about half the distance to pheS, the first structural gene of the operon. The present report shows that, in fact, the only open reading frame to be translated efficiently is the leader peptide itself. The alternative leader peptide and the terminator peptide are both translated at a negligible rate.

Amino Acyl-tRNA Synthetases↗
PubMed1987

AUU-to-AUG mutation in the initiator codon of the translation initiation factor IF3 abolishes translational autocontrol of its own gene (infC) in vivo.

We previously showed that Escherichia coli translation initiation factor IF3 regulates the expression of its own gene infC at the translational level in vivo. Here we create two alterations in the infC gene and test their effects on translational autocontrol of infC expression in vivo by measuring beta-galactosidase activity expressed from infC-lacZ gene fusions under conditions of up to 4-fold derepression or 3-fold repression of infC expression. Replacement of the infC promoter with the trp promoter deletes 120 nucleotides of the infC mRNA 5' to the translation initiation site without affecting autogenous translational control. Mutation of the unusual AUU initiator codon of infC to the more common AUG initiator codon abolishes translation initiation factor IF3-dependent repression and derepression of infC expression in vivo. These results establish the AUU initiator codon of infC as an essential cis-acting element in autogenous translational control of translation initiation factor IF3 expression in vivo.

Bacteriophage lambda↗
PubMed1986

Escherichia coli protein synthesis initiation factor IF3 controls its own gene expression at the translational level in vivo.

Measurements of the relative synthesis rates of mRNAs transcribed from the gene (thrS) for threonyl-tRNA synthetase and the adjacent gene (infC) for initiation factor IF3 show four- to fivefold more infC mRNA than thrS mRNA in vivo, suggesting that infC expression can be controlled independently of thrS expression. S1 mapping experiments reveal the existence of two transcription initiation sites for infC mRNAs internal to the thrS structural gene. Both the mRNA measurements and the S1 mapping experiments indicate that the majority of infC transcription initiates at the infC proximal promoter. In agreement with these results, the deletion of the infC distal promoter from infC-lacZ gene fusions does not affect the expression of these gene fusions in vivo. Measurements of the relative synthesis rate of infC mRNA in vivo in infC- strains overproducing IF3 shows that infC mRNA levels are normal in these strains, thus suggesting that IF3 regulates the translation of infC mRNAs in vivo. Extension of these experiments using infC-lacZ gene fusions carried on lambda bacteriophage and integrated at the lambda att site on the Escherichia coli chromosome shows that the expression of infC-lacZ protein fusions, but not infC-lacZ operon fusions, is derepressed in two infC- strains. A cellular excess of IF3 represses the expression of an infC-lacZ protein fusion but not an infC-lacZ operon fusion. Measurements of the relative mRNA synthesis rates of hybrid infC-lacZ mRNA synthesized from an infC-lacZ protein fusion under conditions of a fourfold derepression or a threefold repression of hybrid IF3-beta-galactosidase expression shows that the hybrid infC-lacZ mRNA levels remain unchanged. These results indicate that the cellular levels of IF3 negatively regulate the expression of its own gene, infC, at the translational level in vivo.

Escherichia coli↗
PubMed1986

Cross-linking of initiation factor IF3 to Escherichia coli 30S ribosomal subunit by trans-diamminedichloroplatinum(II): characterization of two cross-linking sites in 16S rRNA; a possible way of functioning for IF3.

The initiation factor IF3 is platinated with trans-diamminedichloroplatinum(II) and cross-linked to Escherichia coli 30S ribosomal subunit. Two cross-linking sites are unambiguously identified on the 16S rRNA: a major one, in the region 819-859 in the central domain, and a minor one, in the region 1506-1529 in the 3'-terminal domain. Specific features of these sequences together with their particular location within the 30S subunit lead us to postulate a role for IF3, that conciliates topographical and functional observations made so far.

Base Sequence↗
PubMed1986

Altered translation initiation factor 2 in the cold-sensitive ssyG mutant affects protein export in Escherichia coli.

The Escherichia coli gene secY (pr1A) codes for an integral membrane protein that plays an essential role in protein export. We previously isolated cold-sensitive mutations (ssy) as extragenic suppressors of temperature-sensitive secY24 mutation. Now we show that the ssyG class of mutations are within infB coding for the translation initiation factor IF2. The mutants produce altered forms of IF2 with a cold-sensitive in vitro activity to form a translation initiation complex. The mutation suppresses not only secY24 but also other secretion-defective mutations such as secA51 and rp10215. The beta-galactosidase enzyme activity of the MalE-LacZ 72-47 hybrid protein is strikingly reduced in the ssyG mutant at the permissive high temperature, while the hybrid protein itself is normally synthesized. This effect, which was observed only for the hybrid protein with a functional signal sequence, may result from some alteration in the cellular localization of the protein. These results suggest that IF2 or the translation initiation step can modulate protein export reactions. The isolation of cold-sensitive ssyG mutations in infB provides genetic evidence that IF2 is indeed essential for normal growth of E. coli cells.

Bacterial Proteins↗
PubMed1986

Genetic definition of the translational operator of the threonine-tRNA ligase gene in Escherichia coli.

The Escherichia coli gene thrS that codes for threonine-tRNA ligase (tRNAThr ligase, formerly threonine-tRNA synthetase, EC 6.1.1.3) has previously been shown to be negatively autoregulated at the level of translation. Here we describe the use of several thrS-lac gene fusions to isolate cis-acting regulatory mutations that increase the translation but not the transcription of the thrS gene. These mutations lead to a total loss of control of repression and derepression of thrS. DNA sequence analysis locates the mutations between 10 and 40 base pairs upstream of the translation initiation codon of thrS and more than 100 base pairs downstream of the transcription initiation site. The mRNA region where these mutations are located shares primary and secondary structure homologies with specific parts of several isoacceptor tRNAThr species. These findings suggest that the ligase regulates its translation by binding to its mRNA at a place that shares some homology with its natural substrate.

Amino Acyl-tRNA Synthetases↗
PubMed1986

Posttranscriptional autoregulation of Escherichia coli threonyl tRNA synthetase expression in vivo.

Five mutations in thrS, the gene for threonyl-tRNA synthetase, have been characterized, and the sites of the mutations have been localized to different regions of the thrS gene by recombination with M13 phage carrying portions of the thrS gene. Quantitative immunoblotting shows that some of these mutations cause the overproduction of structurally altered threonyl-tRNA synthetase in vivo. The amounts of in vivo thrS mRNA as measured by quantitative hybridization are, however, the same as wild-type levels for each mutant. These results demonstrate that the expression of threonyl-tRNA synthetase is autoregulated at the posttranscriptional level in vivo.

Amino Acyl-tRNA Synthetases↗
PubMed1985

Autogenous control of Escherichia coli threonyl-tRNA synthetase expression in vivo.

The regulation of the expression of thrS, the structural gene for threonyl-tRNA synthetase, was studied using several thrS-lac fusions cloned in lambda and integrated as single copies at att lambda. It is first shown that the level of beta-galactosidase synthesized from a thrS-lac protein fusion is increased when the chromosomal copy of thrS is mutated. It is also shown that the level of beta-galactosidase synthesized from the same protein fusion is decreased if wild-type threonyl-tRNA synthetase is overproduced from a thrS-carrying plasmid. These results strongly indicate that threonyl-tRNA synthetase controls the expression of its own gene. Consistent with this hypothesis it is shown that some thrS mutants overproduce a modified form of threonyl-tRNA synthetase. When the thrS-lac protein fusion is replaced by several types of thrS-lac operon fusions no effect of the chromosomal thrS allele on beta-galactosidase synthesis is observed. It is also shown that beta-galactosidase synthesis from a promoter-proximal thrS-lac operon fusion is not repressed by threonyl-tRNA synthetase overproduction. The fact that regulation is seen with a thrS-lac protein fusion and not with operon fusions indicates that thrS expression is autoregulated at the translational level. This is confirmed by hybridization experiments which show that under conditions where beta-galactosidase synthesis from a thrS-lac protein fusion is derepressed three- to fivefold, lac messenger RNA is only slightly increased.

Amino Acyl-tRNA Synthetases↗
PubMed1985

Control of phenylalanyl-tRNA synthetase genetic expression. Site-directed mutagenesis of the pheS, T operon regulatory region in vitro.

Previous studies of phenylalanyl-tRNA synthetase expression in Escherichia coli strongly suggested that the pheS, T operon was regulated by a phenylalanine-mediated attenuation mechanism. To investigate the functions of the different segments composing the pheS, T attenuator site, a series of insertion, deletion and point mutations in the pheS, T leader region have been constructed in vitro on a recombinant M13 phage. The effects of these alterations on the regulation of the operon were measured after transferring each mutation onto a lambda phage carrying a pheS, T-lacZ fusion. The behaviours of the various mutants agree with the predictions of the attenuation model. The role of the antiterminator (2-3 pairing) as competitor of the terminator (3-4 pairing) is demonstrated by several mutations affecting the stability of the 2-3 base-pairing. The existence of deletions and point mutations in the 3-4 base-pairing shows that the terminator is essential for both expression level and regulation of the operon. Mutations in the translation initiation site of the leader peptide show that the expression of the leader peptide is essential for attenuation control. However, alteration of the translation initiation rate of the leader peptide derepresses the pheS, T operon, which is the opposite of what is observed with the trp operon. This difference is explained in terms of different translation initiation efficiencies of the leader peptides. Finally, insertion mutations, increasing gradually the distance between the leader peptide stop codon and the first strand of the antiterminator, derepress the pheS, T operon and show that formation of the antiterminator structure is under the control of the translation of the leader peptide.

Amino Acyl-tRNA Synthetases↗
PubMed1985

Effect of NusA protein on expression of the nusA,infB operon in E. coli.

Protein and operon fusions between lacZ and various genes of the nusA,infB operon have been constructed on lambda bacteriophages and used to show that the operon is negatively regulated by the level of NusA protein. Overproducing NusA (but not IF2) from a multicopy plasmid reduces the level of beta-galactosidase from the fusions indicating repression of the operon. Introducing the lambda carrying the fusions into nusA mutant strains produces a higher level of beta-galactosidase-indicative of derepression of the operon. In particular, a larger form of the NusA protein which does not affect bacterial growth per se causes a derepression of the operon. As both protein and operon fusions respond equivalently, we conclude that the nusA protein is acting at the transcriptional level to regulate expression of the nusA, infB operon.

Bacterial Proteins↗
PubMed1985

Attenuation control of the Escherichia coli phenylalanyl-tRNA synthetase operon.

The pheST operon codes for the two subunits of the essential enzyme phenylalanyl-tRNA synthetase. The nucleotide sequence of the regulatory regions of the operon, in vitro transcription data and in vivo experiments indicate that the operon is controlled by attenuation in a way similar to many amino acid biosynthetic operons. In this work the control of the pheST operon was studied in vivo by measuring the effect of deletions in the regulatory regions on downstream expression. The presence of a strong promoter followed by an approximately 90% efficient terminator in front of the structural parts of the operon is demonstrated. An open reading frame coding for a 14 amino acid long leader peptide containing five phenylalanine residues is located between the promoter and the terminator. The presence of the transcription terminator is shown to be essential to the operon's regulation. The localization of the promoter and the terminator agrees with the results of previous in vitro experiments. It is also shown that about 30% of the transcripts covering the pheST operon come from the upstream gene, rplT, which codes for the ribosomal protein L20. Although cotranscription exists between rplT and pheST, these genes are not systematically coregulated since reducing the translation of rplT about tenfold, does not change pheST expression. The pheST operon is also shown to be derepressed by a cellular excess of phenylalanyl-tRNA synthetase. This derepression is shown to be due to the pheST attenuator.

Amino Acyl-tRNA Synthetases↗
PubMed1985

Two translational initiation sites in the infB gene are used to express initiation factor IF2 alpha and IF2 beta in Escherichia coli.

The gene infB codes for the two forms of translational initiation factor IF2: IF2 alpha (97 300 daltons) and IF2 beta (79 700 daltons). To determine whether the two forms differ at their N terminus, purified IF2 alpha and IF2 beta were subjected to 11 or more steps of Edman degradation. The N-terminal amino acid sequences are completely different, but match perfectly the DNA sequences at the beginning of the infB open reading frame and an in-phase region 471 bp downstream. A fusion was constructed between the proximal half of the infB gene and the lacZ gene lacking the region coding for the first eight amino acids. The fused gene expresses two products of 170 000 and 150 000 daltons, corresponding to the fused proteins IF2 alpha-beta-galactosidase and IF2 beta-beta-galactosidase, which confirms in vivo that the IF2 forms differ at their N terminus. A deletion of the 5'-non-translated region of the fused gene, including the Shine/Dalgarno ribosomal binding site, results in the expression of IF2 beta-beta-galactosidase but not IF2 alpha-beta-galactosidase. This strongly suggests that IF2 beta results from independent translation rather than from a precise proteolytic cleavage of IF2 alpha. Further evidence for initiation of protein synthesis at the putative IF2 alpha and IF2 beta start sites was sought by using an in vitro dipeptide synthesis assay. A DNA fragment containing the entire infB gene was cloned into three plasmid vectors and the resulting recombinant DNAs were used as templates in assays containing fMet-tRNA and various labelled aminoacyl-tRNAs.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1985

Evidence for autoregulation of the nusA-infB operon of Escherichia coli.

Analysis of three different nusA mutant strains suggests that the expression of the nusA-infB operon of Escherichia coli is regulated autogenously by the nusA gene product, a protein known to mediate transcription termination and antitermination. The cellular amounts of NusA and IF2 (infB) proteins are enhanced by a nusAts mutation which causes reduced transcription-termination activity. A nusAam mutant carrying the am ts suppressor, supFts6, overproduces the IF2 protein when the amount of NusA protein is reduced by the thermal inactivation of the supFts6. A modified form of NusA with the cat protein of Mr of 24 000 attached to the C terminus of NusA is overproduced compared to the wild-type NusA and causes the overproduction of IF2.

Bacterial Proteins↗
PubMed1985

Overproduction and purification of initiation factor IF2 and pNUSA proteins from a recombinant plasmid bearing strain.

The genes for translational initiation factor, IF2 and pNusA have been cloned into a plasmid vector where they are placed under the control of the inducible lambdapL promoter and the c1857 thermosensitive repressor. When a strain carrying this plasmid is heat induced, IF2 alpha, IF2 beta and pNusA are overproduced 15 to 20 fold. This has allowed us to purify the IF2 and NusA proteins in large amounts.

Bacterial Proteins↗