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M Grunberg-Manago

Publications and source records attributed to M Grunberg-Manago.

At least 55 records · Page 3Linked to original sources
PubMed1990

Target site of Escherichia coli ribosomal protein S15 on its messenger RNA. Conformation and interaction with the protein.

The regulatory site of ribosomal protein S15 has been located in the 5' non-coding region of the messenger, overlapping with the ribosome loading site. The conformation of an in vitro synthesized mRNA fragment, covering the 105 nucleotides upstream from the initiation codon and the four first codons of protein S15, has been monitored using chemical probes and RNase V1. Our results show that the RNA is organized into three domains. Domains I and II, located in the 5' part of the mRNA transcript, are folded into stable stem-loop structures. The 3'-terminal domain (III), which contains the Shine-Dalgarno sequence and the AUG initiation codon, appears to adopt alternative conformations. One of them corresponds to a rather unstable stem-loop structure in which the Shine-Dalgarno sequence is paired. An alternative potential structure involves a "pseudo-knot" interaction between bases of this domain and bases in the loop of domain II. The conformation of several RNA variants has also been investigated. The deletion of the 5'-proximal stem-loop structure (domain I), which has no effect on the regulation, does not perturb the conformation of the two other domains. The deletion of domain II, leading to a loss of regulatory control, prevents the formation of the potential helix involved in the pseudo-knot structure and results in a stabilization of the alternative stem-loop structure in domain III. The replacement of another base in domain III involved in pairing in the two alternative structures mentioned above should induce a destabilization of both structures and results in a loss of the translational control. However, the replacement of another base in domain III, which does not abolish the control, results in the loss of the conformational heterogeneity in this domain and yields a stable conformation corresponding to the pseudo-knot structure. Thus, it appears that any mutation that disrupts or alters the formation of the pseudo-knot impairs the regulatory mechanism. Footprinting experiments show that protein S15 is able to bind to the synthesized fragment and provide evidence that the protein triggers the formation of the pseudo-knot conformation. A mechanism can be postulated in which the regulatory protein stabilizes this particular structure, thus impeding ribosome initiation.

Base Composition↗
PubMed1990

RNase III cleavages in non-coding leaders of Escherichia coli transcripts control mRNA stability and genetic expression.

The primary transcripts of the rpsO-pnp, rnc-era-recO and metY-nusA-infB operons of E coli are each processed by RNase III, upstream of the first translated gene, in hair-pin structures formed by the 5' non-coding leader. The mRNAs of the 3 operons, of which the 5' terminal motifs have been removed by RNase III, decay significantly more rapidly than the uncut transcripts which accumulate in the RNase III deficient strain. The rapid decay of a primary transcript of the metY-nusA-infB operon, initiated at a secondary promoter in the vicinity of the RNase III sites, suggests that the 5' features upstream of the RNase III cutting sites are responsible for the stability of the uncut RNAs. RNase III autocontrols its own expression by removing the 5' motif which stabilizes its mRNA. Similarly, the synthesis of polynucleotide phosphorylase and of protein Era are also controlled by RNase III cleavages which trigger the degradation of their messengers. The role of RNase III in the regulation of gene expression and the possible mechanisms of mRNA stabilization and of 5' to 3' decay initiated by RNase III processing are discussed.

Base Sequence↗
PubMed1990

Isolation and molecular genetic characterization of the Bacillus subtilis gene (infB) encoding protein synthesis initiation factor 2.

Western blot (immunoblot) analysis of Bacillus subtilis cell extracts detected two proteins that cross-reacted with monospecific polyclonal antibody raised against Escherichia coli initiation factor 2 alpha (IF2 alpha). Subsequent Southern blot analysis of B. subtilis genomic DNA identified a 1.3-kilobase (kb) HindIII fragment which cross-hybridized with both E. coli and Bacillus stearothermophilus IF2 gene probes. This DNA was cloned from a size-selected B. subtilis plasmid library. The cloned HindIII fragment, which was shown by DNA sequence analysis to encode the N-terminal half of the B. subtilis IF2 protein and 0.2 kb of upstream flanking sequence, was utilized as a homologous probe to clone an overlapping 2.76-kb ClaI chromosomal fragment containing the entire IF2 structural gene. The HindIII fragment was also used as a probe to obtain overlapping clones from a lambda gt11 library which contained additional upstream and downstream flanking sequences. Sequence comparisons between the B. subtilis IF2 gene and the other bacterial homologs from E. coli, B. stearothermophilus, and Streptococcus faecium displayed extensive nucleic acid and protein sequence homologies. The B. subtilis infB gene encodes two proteins, IF2 alpha (78.6 kilodaltons) and IF2 beta (68.2 kilodaltons); both were expressed in B. subtilis and E. coli. These two proteins cross-reacted with antiserum to E. coli IF2 alpha and were able to complement in vivo an E. coli infB gene disruption. Four-factor recombination analysis positioned the infB gene at 145 degrees on the B. subtilis chromosome, between the polC and spcB loci. This location is distinct from those of the other major ribosomal protein and rRNA gene clusters of B. subtilis.

Amino Acid Sequence↗
PubMed1990

Independent genes for two threonyl-tRNA synthetases in Bacillus subtilis.

With the exception of Escherichia coli lysyl-tRNA synthetase, the genes coding for the different aminoacyl-tRNA synthetases in procaryotes are always unique. Here we report on the occurrence and cloning of two genes (thrSv and thrS2), both encoding functional threonyl-tRNA synthetase in Bacillus subtilis. The two proteins share only 51.5% identical residues, which makes them almost as distinct from each other as each is from E. coli threonyl-tRNA synthetase (42 and 47%). Both proteins complement an E. coli thrS mutant and effectively charge E. coli threonyl tRNA in vitro. Their genes have been mapped to 250 degrees (thrSv) and 344 degrees (thrS2) on the B. subtilis chromosome. The regulatory regions of both genes are quite complex and show structural similarities. During vegetative growth, only the thrSv gene is expressed.

Amino Acid Sequence↗
PubMed1989

The solution structure of the Escherichia coli initiator tRNA and its interactions with initiation factor 2 and the ribosomal 30 S subunit.

The conformation of the Escherichia coli initiator tRNA has been investigated using enzymatic and chemical probes. This study was conducted on the naked tRNA and on the tRNA involved in the various steps leading to the formation of the 30 S.IF-2.GTP.fMet-tRNA.AUG complex. A three-dimensional model of the initiator tRNA is presented, which displays several differences with yeast tRNAPhe: (i) the anticodon arm is more rigid; (ii) the presence of an additional nucleotide in the D loop results in specific features in both T and D loops; (iii) C1 and A72 might form a noncanonical base pair. Aminoacylation and formylation induce subtle conformational adjustments near the 3' end, the T arm and the D loop. Initiation factor (IF) 2 interacts with a rather limited portion of the tRNA, covering the T loop and the minor groove of the T stem, and induces an increased flexibility in the anticodon arm. The specific structural features observed in the T loop are probably recognized by IF-2. In the 30 S.IF-2.GTP.fMet-tRNA.AUG complex, additional protections are observed in the acceptor stem and in the anticodon arm, resulting from a strong steric hindrance and from the codon-anticodon interaction within the subunit decoding site.

Base Sequence↗
PubMed1989

Cleavage by RNase III in the transcripts of the met Y-nus-A-infB operon of Escherichia coli releases the tRNA and initiates the decay of the downstream mRNA.

The metY gene coding for a minor form of the initiator tRNA is the first gene of a complex polycistronic operon also encoding the transcription termination factor NusA and the translation initiation factor IF2. The mixed tRNA-mRNA polycistronic transcript is cleaved by RNase III in a hairpin structure downstream from the tRNA. This cleavage separates the tRNA from the mRNA and initiates the rapid degradation of the 5' extremity of the downstream mRNA. Dissociation of the structural (tRNA) and informational (mRNA) RNAs from this operon is also achieved by independent transcription in vivo. The presence of two transcription terminators located downstream from metY produces a small tRNAMetf2 precursor transcript, whereas an internal promoter situated between metY and the first open reading frame directs the transcription of only the protein-coding part of the operon.

Bacterial Proteins↗
PubMed1989

tRNA-like structures and gene regulation at the translational level: a case of molecular mimicry in Escherichia coli.

Escherichia coli threonyl-tRNA synthetase regulates the translation of its own mRNA by binding to it in a region, called the operator, located in front of the ribosomal binding site. The primary and secondary structures of the operator resemble those of the anticodon arm of several tRNA(Thr) isoacceptor species. We reasoned that if the interaction between the synthetase and its two partially analogous ligands, the tRNA and the mRNA, had some common features, single mutations in the enzyme should affect both interactions in a very similar way. We thus isolated synthetase mutants (called super-repressors) that repress the translation of their mRNA in trans to an extreme level, and other mutants that are completely unable to perform any repression. The super-repressors, which are suspected to bind their mRNA with high affinity, are shown to bind the tRNA with an increased affinity. The non-repressing mutants, which are suspected to have lost their capacity to bind the mRNA, are shown to bind their tRNA with less affinity. The binding properties of the mutant enzymes for the other substrates, ATP and threonine, are unchanged. The observed correlation between regulatory and aminoacylation defects strongly suggests that the synthetase recognizes the similar parts of its two RNA ligands--the anticodon-like arm of the mRNA and the true anticodon arm of the tRNA--in an analogous way.

Amino Acid Sequence↗
PubMed1989

Autoregulation of RNase III operon by mRNA processing.

RNase III has been implicated in the control of gene expression by the processing of mRNA. We have found that the rnc operon is autoregulated; rnc- mutant strains oversynthesize the operon's mRNA and protein products. A site in the 5'-noncoding region of the operon's message is cleaved by RNase III. This site-specific cleavage appears to be the initial step in the functional inactivation of the message, since the half-life of the cut message is dramatically shorter than that of the uncut message.

Base Sequence↗
PubMed1989

Bacillus subtilis phenylalanyl-tRNA synthetase genes: cloning and expression in Escherichia coli and B. subtilis.

The genes that encode the two subunits of Bacillus subtilis phenylalanyl-tRNA synthetase were cloned from alpha lambda library of chromosomal B. subtilis DNA by specific complementation of a thermosensitive Escherichia coli pheS mutation. Both genes (we named them pheS and pheT, analogous to the corresponding genes of E. coli) are carried by a 6.6-kilobase-pair PstI fragment which also complements E. coli pheT mutations. This fragment directs the synthesis of two proteins identical in size to the purified alpha and beta subunits of the phenylalanyl-tRNA synthetase of B. subtilis with Mrs of 42,000 and 97,000, respectively. A recombinant shuttle plasmid carrying the genes caused 10-fold overproduction of functional phenylalanyl-tRNA synthetase in B. subtilis.

Amino Acyl-tRNA Synthetases↗
PubMed1988

The existence of two genes between infB and rpsO in the Escherichia coli genome: DNA sequencing and S1 nuclease mapping.

A number of genes encoding proteins involved in transcription and translation are clustered between 68 and 69 minutes on the Escherichia coli genome map and are transcribed clockwise as two operons: the metY operon, containing metY, P15A, nusA, infB; and about a kilobase further downstream, the rpsO and pnp operon. The DNA sequence between infB and rpsO was determined and two open reading frames were detected which code for proteins of 15,200 (P15B) and 35,091 (P35) daltons. Maxicell analysis showed a relatively strong expression of P15B whereas P35 was synthesized more weakly. An overlap of the termination codon of P15B and the initiator codon for P35 suggests that translation of P15B and P35 may be coupled. S1 nuclease mapping of in vivo transcripts between infB and rpsO provided no evidence for major promoters but detected a moderately efficient rho-independent terminator between infB and P15B. The results indicate that P15B and P35 are expressed as part of the metY operon, but that some transcriptional read through into the rpsO operon also occurs, thereby, functionally linking the expression of these two complex systems.

Amino Acid Sequence↗
PubMed1988

Tertiary structure of Escherichia coli tRNA(3Thr) in solution and interaction of this tRNA with the cognate threonyl-tRNA synthetase.

The solution structure of Escherichia coli tRNA(3Thr) (anticodon GGU) and the residues of this tRNA in contact with the alpha 2 dimeric threonyl-tRNA synthetase were studied by chemical and enzymatic footprinting experiments. Alkylation of phosphodiester bonds by ethylnitrosourea and of N-7 positions in guanosines and N-3 positions in cytidines by dimethyl sulphate as well as carbethoxylation of N-7 positions in adenosines by diethyl pyrocarbonate were conducted on different conformers of tRNA(3Thr). The enzymatic structural probes were nuclease S1 and the cobra venom ribonuclease. Results will be compared to those of three other tRNAs, tRNA(Asp), tRNA(Phe) and tRNA(Trp), already mapped with these probes. The reactivity of phosphates towards ethylnitrosourea of the unfolded tRNA was compared to that of the native molecule. The alkylation pattern of tRNA(3Thr) shows some similarities to that of yeast tRNA(Phe) and mammalian tRNA(Trp), especially in the D-arm (positions 19 and 24) and with tRNA(Trp), at position 50, the junction between the variable region and the T-stem. In the T-loop, tRNA(3Thr), similarly to the three other tRNAs, shows protections against alkylation at phosphates 59 and 60. However, tRNA(3Thr) is unique as far as very strong protections are also found for phosphates 55 to 58 in the T-loop. Compared with yeast tRNA(Asp), the main differences in reactivity concern phosphates 19, 24 and 50. Mapping of bases with dimethyl sulphate and diethyl pyrocarbonate reveal conformational similarities with yeast tRNA(Phe). A striking conformational feature of tRNA(3Thr) is found in the 3'-side of its anticodon stem, where G40, surrounded by two G residues, is alkylated under native conditions, in contrast to other G residues in stem regions of tRNAs which are unreactive when sandwiched between two purines. This data is indicative of a perturbed helical conformation in the anticodon stem at the level of the 30-40 base pairs. Footprinting experiments, with chemical and enzymatic probes, on the tRNA complexed with its cognate threonyl-tRNA synthetase indicate significant protections in the anticodon stem and loop region, in the extra-loop, and in the amino acid accepting region. The involvement of the anticodon of tRNA(3Thr) in the recognition process with threonyl-tRNA synthetase was demonstrated by nuclease S1 mapping and by the protection of G34 and G35 against alkylation by dimethyl sulphate. These data are discussed in the light of the tRNA/synthetase recognition problem and of the structural and functional properties of the tRNA-like structure present in the operator region of the thrS mRNA.

Affinity Labels↗
PubMed1988

Translational control in E. coli: the case of threonyl-tRNA synthetase.

Genetic studies have shown that expression of the E. coli threonyl-tRNA synthetase (thrS) gene is negatively auto-regulated at the translational level. A region called the operator, located 110 nucleotides downstream of the 5' end of the mRNA and between 10 and 50 bp upstream of the translational initiation codon in the thrS gene, is directly involved in that control. The conformation of an in vitro RNA fragment extending over the thrS regulatory region has been investigated with chemical and enzymatic probes. The operator locus displays structural similarities to the anti-codon arm of threonyl tRNA. The conformation of 3 constituent mutants containing single base changes in the operator region shows that replacement of a base in the anti-codon-like loop does not induce any conformational change, suggesting that the residue concerned is directly involved in regulation. However mutation in or close to the anti-codon-like stem results in a partial or complete rearrangement of the structure of the operator region. Further experiments indicate that there is a clear correlation between the way the synthetase recognises each operator, causing translational repression, and threonyl-tRNA.

Amino Acyl-tRNA Synthetases↗
PubMed1988

Messenger RNA structure and gene regulation at the translational level in Escherichia coli: the case of threonine:tRNAThr ligase.

Previous work showed that the expression of the Escherichia coli threonine:tRNAThr ligase (EC 6.1.1.3)-encoding gene (thrS) is negatively autoregulated at the translational level and that a region called the operator that is located between 10 and 50 base pairs upstream of the translation initiation codon of the thrS gene is directly involved in that control. The conformation of an in vitro synthesized RNA fragment extending over the thrS regulatory region has been investigated using chemical and enzymatic probes. This study shows that the RNA folds into four well-defined secondary-structure domains, one of them displaying structural similarities to the anticodon arm of tRNAThr. The conformation of three constitutive mutants containing single base changes in the operator region leading to the loss of the regulatory control was also investigated. The replacement of a base in the anticodon-like loop does not induce any conformational change, suggesting that the residue concerned is directly involved in the regulatory process. However, single mutations in or close to the anticodon-like stem result in a partial or complete reorganization of the structure of the operator region. These rearrangements should affect the binding of the ligase to the operator, leading to loss of the regulatory process.

Amino Acyl-tRNA Synthetases↗
PubMed1987

Feedback regulation of rRNA synthesis in Escherichia coli. Requirement for initiation factor IF2.

It has been shown that the transcription of rRNA in Escherichia coli is feedback-regulated by its own transcription products through a negative feedback loop which appears to require the assembly of rRNA into complete ribosomes. In order to examine whether the feedback loop involves the ribosomes' main function, translation, we have constructed a strain in which the chromosomal copy of infB, encoding IF2, was placed under lac promoter/operator control, and the effects of limitation of translation initiation factor IF2 on the regulation were examined. By varying the concentration of a lac operon inducer, isopropyl thiogalactoside (IPTG), it was possible to vary the cellular concentration of IF2. Under the growth conditions used, decreasing the concentration of IF2 about twofold affected the growth rate only slightly, but further deprivation of IF2 resulted in a significant decrease in growth rate, an increase in RNA content and a large accumulation of non-translating ribosomes. These accumulated ribosomes were apparently unable to cause feedback regulation of rRNA synthesis in the absence of sufficient IF2. When a higher concentration of IPTG was added to these IF2-deficient cells, a rapid increase in the IF2 level and a significant decrease in the rate of RNA accumulation were observed before the new steady-state growth was attained. These results indicate that IF2 apparently is necessary for feedback regulation of stable RNA and imply that ribosomes must enter translation for feedback regulation to occur.

Centrifugation, Density Gradient↗
PubMed1987

The protein synthesis initiation factor 2 G-domain. Study of a functionally active C-terminal 65-kilodalton fragment of IF2 from Escherichia coli.

Protein synthesis initiation factor 2 (IF2) is present in Escherichia coli cells as two forms which are expressed from the same gene: IF2 alpha [97.3 kilodaltons (kDa)] and IF2 beta (79.7 kDa). During isolation, a smaller form, IF2 gamma, is generated, presumably by partial proteolysis. It has been purified to homogeneity and has an apparent mass of 70 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoelectrophoresis of IF2 alpha and IF2 gamma shows that IF2 gamma is immunologically partially identical with IF2 alpha. The sequence of the 15 N-terminal amino acid residues of IF2 gamma was determined and compared with that of IF2 alpha. The N-terminal amino acid of IF2 gamma corresponds to Arg-290 of IF2 alpha, suggesting that IF2 gamma is generated by proteolytic cleavage of the Lys-289-Arg-290 bond of IF2. Assuming a C terminus identical with IF2 alpha, we calculate that IF2 gamma comprises 601 amino acid residues and has a mass of 64.8 kDa. The truncated protein was tested for activities characteristic of IF2 in three in vitro assays: fMet-tRNA(fMet) binding to 70S ribosomes, N-terminal dipeptide synthesis in a DNA-dependent transcription/translation system, and ribosome-dependent GTP hydroly97-7. The specific activities of IF2 gamma were comparable with, or only slightly less than, those for IF2 alpha, indicating that IF2 gamma contains the active centers for interaction with fMet-tRNA(fMet), ribosomes, and GTP. A central region in the primary structure of IF2 shows extensive sequence homology with a number of GDP-binding proteins and especially with the G-domain of elongation factor Tu (EF-Tu).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗