Growth rate-dependent control, feedback regulation and steady-state mRNA levels of the threonyl-tRNA synthetase gene of Escherichia coli.

The expression of the gene thrS encoding threonyl-tRNA synthetase is under the control of two apparently different regulatory loops: translational feedback regulation and growth rate-dependent control. The translational feedback regulation is due to the binding of threonyl-tRNA synthetase to a site located in the leader RNA of thrS, upstream of the initiation codon, which mimics the anticodon stem and loop of tRNA(Thr). This binding competes with that of the ribosome and thus inhibits translation initiation. Here, we investigate the mechanism of growth rate-dependent control, i.e. the mechanism by which the synthetase accumulates at high growth rates. We show that growth rate-dependent control acts at the level of translation and requires feedback regulation since mutations that abolish feedback regulation also abolish growth rate-dependent control. We also show that tRNA(Thr), which accumulates at high growth rates, is one of the effectors of growth rate-dependent control since its accumulation can cause derepression independently of growth rate. We show that this tRNA(Thr)-dependent derepression is also dependent on feedback regulation since mutations which abolish feedback also prevent derepression. Based on these results and previous data concerning the mechanism of translational feedback regulation, we propose that threonyl-tRNA synthetase growth rate-dependent control is the consequence of the accumulation at high growth rates of two effectors, the ribosome and tRNA(Thr). We also study the growth rate-dependence of the steady state level of thrS mRNA and show that the steady state level of thrS mRNA increases at high growth rates. This increase is dependent on the translational feedback regulation and can also be detected, independently of growth rate, when thrS mRNA translation is derepressed. Consistently with the model of growth rate-dependent control above, we propose that at high growth rates, the mRNA is well translated and thus stabilised and that, at low growth rates, because of its low translation, thrS mRNA is rapidly degraded.

The role of the AUU initiation codon in the negative feedback regulation of the gene for translation initiation factor IF3 in Escherichia coli.

The expression of the infC gene encoding translation initiation factor IF3 is negatively autoregulated at the level of translation, i.e. the expression of the gene is derepressed in a mutant infC background where the IF3 activity is lower than that of the wild type. The special initiation codon of infC, AUU, has previously been shown to be essential for derepression in vivo. In the present work, we provide evidence that the AUU initiation codon causes derepression by itself, because if the initiation codon of the thrS gene, encoding threonyl-tRNA synthetase, is changed from AUG to AUU, its expression is also derepressed in an infC mutant background. The same result was obtained with the rpsO gene encoding ribosomal protein S15. We also show that derepression of infC, thrS, and rpsO is obtained with other 'abnormal' initiation codons such as AUA, AUC, and CUG which initiate with the same low efficiency as AUU, and also with ACG which initiates with an even lower efficiency. Under conditions of IF3 excess, the expression of infC is repressed in the presence of the AUU or other 'abnormal' initiation codons. Under the same conditions and with the same set of 'abnormal' initiation codons, the repression of thrS and rpsO expression is weaker. This result suggests that the infC message has specific features that render its expression particularly sensitive to excess of IF3. We also studied another peculiarity of the infC message, namely the role of a GC-rich sequence located immediately downstream of the initiation codon and conserved through evolution. This sequence was proposed to interact with a conserved region in 16S RNA and enhance translation initiation. Unexpectedly, mutating this GC-rich sequence increases infC expression, indicating that this sequence has no enhancing role. Chemical and enzymatic probing of infC RNA synthesized in vitro indicates that this GC-rich sequence might pair with another region of the mRNA. On the basis of our in vivo results we propose, as suspected from earlier in vitro results, that IF3 regulates the expression of its own gene by using its ability to differentiate between 'normal' and 'abnormal' initiation codons.

Stabilised secondary structure at a ribosomal binding site enhances translational repression in E. coli.

The expression of the gene encoding Escherichia coli threonyl-tRNA synthetase is negatively autoregulated at the translational level. The negative feedback is due to the binding of the synthetase to an operator site on its own mRNA located upstream of the initiation codon. The present work describes the characterisation of operator mutants that have the rare property of enhancing repression. These mutations cause (1) a low basal level of expression, (2) a temperature-dependent expression, and (3) an increased capacity of the synthetase to repress its own expression at low temperature. Surprisingly, this enhancement of repression is not explained by an increase of affinity of the mutant operators for the enzyme but by the formation, at low temperature, of a few supplementary base-pairs between the ribosomal binding site and a normally single-stranded domain of the operator. Although this additional base-pairing only slightly inhibits ribosome binding in the absence of repressor, simple thermodynamic considerations indicate that this is sufficient to increase repression. This increase is explained by the competition between the ribosome and repressor for overlapping regions of the mRNA. When the ribosomal binding site is base-paired, the ribosome cannot bind while the repressor can, giving the repressor the advantage in the competition. Thus, the existence of an open versus base-paired equilibrium in a ribosomal binding site of a translational operator amplifies the magnitude of control. This molecular amplification device might be an essential component of translational control considering the low free repressor/ribosome ratio of the low affinity of translational repressors for their target operators.

Messenger RNA secondary structure and translational coupling in the Escherichia coli operon encoding translation initiation factor IF3 and the ribosomal proteins, L35 and L20.

The Escherichia coli infC-rpmI-rplT operon encodes translation initiation factor IF3 and the ribosomal proteins, L35 and L20, respectively. The expression of the last cistron (rplT) has been shown to be negatively regulated at a post-transcriptional level by its own product, L20, which acts at an internal operator located within infC. The present work shows that L20 directly represses the expression of rpmI, and indirectly that of rplT, via translational coupling with rpmI. Deletions and an inversion of the coding region of rpmI, suggest an mRNA secondary structure forming between sequences within rpmI and the translation initiation site of rplT. To verify the existence of this structure, detailed analyses were performed using chemical and enzymatic probes. Also, mutants that uncoupled rplT expression from that of rpmI, were isolated. The mutations fall at positions that would base-pair in the secondary structure. Our model is that L20 binds to its operator within infC and represses the translation of rpmI. When the rpmI mRNA is not translated, it can base-pair with the ribosomal binding site of rplT, sequestering it, and abolishing rplT expression. If the rpmI mRNA is translated, i.e. covered by ribosomes, the inhibitory structure cannot form leaving the translation initiation site of rplT free for ribosomal binding and for full expression. Although translational coupling in ribosomal protein operons has been suspected to be due to the formation of secondary structures that sequester internal ribosomal binding sites, this is the first time that such a structure has been shown to exist.

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.

The specificity of translational control switched with transfer RNA identity rules.

The interaction of Escherichia coli threonyl-transfer RNA (tRNA) synthetase with the leader sequence of its own messenger RNA inhibits ribosome binding, resulting in negative translational feedback regulation. The leader sequence resembles the substrate (tRNA(Thr)) of the enzyme, and the nucleotides that mediate the correct recognition of the leader and the tRNA may be the same. A mutation suggested by tRNA identity rules that switches the resemblance of the leader sequence from tRNA(Thr) to tRNA(Met) causes the translation of the threonyl-tRNA synthetase messenger RNA to become regulated by methionyl-tRNA synthetase. This identity swap in the leader messenger RNA indicates that tRNA identity rules may be extended to interactions of synthetases with other RNAs.

Translated translational operator in Escherichia coli. Auto-regulation in the infC-rpmI-rplT operon.

The genes coding for translation initiation factor IF3 (infC) and for the ribosomal proteins L35 (rpmI) and L20 (rplT) are transcribed in that order from a promoter in front of infC. The last two cistrons of the operon (rpmI and rplT) can be transcribed from a weak secondary promoter situated within the first cistron (infC). Previous experiments have shown that the expression of infC, the first cistron of the operon, is negatively autoregulated at the translational level and that the abnormal AUU initiation codon of infC is responsible for the control. We show that the expression of the last cistron (rplT) is also autoregulated at the posttranscriptional level. The L20 concentration regulates the level of rplT expression by acting in trans at a site located within the first cistron (infC) and thus different from that at which IF3 is known to act. This regulatory site, several hundred nucleotides upstream from the target gene (rplT), was identified through deletions, insertions and a point mutation. Thus, the expression of the operon is controlled in trans by the products of two different cistrons acting at two different sites. The localization within an open reading frame (infC) of a regulatory site acting in cis on the translation of a downstream gene (rplT) is new and was unforeseen since ribosomes translating through the regulatory site might be expected to impair either the binding of L20 or the mRNA secondary structure change caused by the binding. The possible competition between translation of the regions acting in cis and the regulation of the expression of the target gene is discussed.

The relation between catalytic activity and gene regulation in the case of E coli threonyl-tRNA synthetase.

The expression of the gene for threonyl-tRNA synthetase (thrS) has previously been shown as being negatively autoregulated at the translational level. The region of the thrS leader mRNA responsible for that control is located immediately upstream of the ribosomal binding site, and was proposed to fold in a tRNA(Thr) anticodon arm-like structure. The present paper reviews experiments using enzymatic and chemical probes that prove the existence of a tRNA(Thr) anticodon-like structure in the thrS mRNA. These structural studies have also shown the presence of another arm upstream in the leader mRNA that has striking similarities with the acceptor arm of the tRNA(Thr) isoacceptors. This second arm was shown, by mutational analysis, to also be involved in thrS regulation. Footprinting experiments have shown that both the anticodon-like and the acceptor-like arms interact with the synthetase. Finally, the similarity of the interaction of the synthetase with its 2 RNA ligands (mRNA and tRNA) has been investigated by selecting and studying mutants of the synthetase itself. The observed correlation between regulatory and aminoacylation defects in these mutants strongly suggests that the synthetase recognizes similar regions of its 2 RNA ligands in an analogous manner.

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.

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.

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)

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.

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.

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.

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.

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.

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.

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.

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.

Expression of the gene for Escherichia coli initiation factor IE-3 in vivo and in vitro.

Expression of protein synthesis initiation factor IF-3 in vivo was studied by measuring its level in exponentially growing cells as a function of gene dosage. A strain haploid for infC, the gene for IF-3, was modified to carry one or two additional infC genes giving diploid and triploid strains. Polyploid strains were achieved by the presence of multicopy plasmids expressing the infC gene. When IF-3 levels were measured by quantitative immunoblotting they were found to be proportional to the gene dosage; the presence of a multicopy plasmid thus causes considerable overproduction of IF-3, enabling large quantities to be purified. When lysates were prepared from freshly grown cells, only IF-3 alpha (the long form) was detected; however when IF-3 was purified from a strain containing a multicopy plasmid which overproduced it, the major product found was IF-3 beta (the short form, lacking six amino acids from the N terminus). The synthesis of the two IF-3 forms was also studied by using a cell-free coupled transcription-translation system dependent on exogenous DNA: the IF-3 gene was found to be very efficiently expressed. IF-3 alpha increased more rapidly than IF-3 beta but following the cessation of protein synthesis IF-3 alpha decreased while IF-3 beta still increased. The results suggest that IF-3 alpha is slowly degraded to the beta form. Addition of non-radioactive IF-3 alpha, up to fivefold molar excess over ribosomes, to the synthesizing system in vitro did not inhibit IF-3 synthesis. Synthesis of IF-3 in vitro appears to be sensitive to guanosine 3'-diphosphate 5'-diphosphate.