M. Graffe

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 infCthrS, 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.

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.

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.

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.

IF-3 requirements for initiation complex formation with synthetic messengers in E. coli system.

The polypeptide chain initiation factor IF-3, which participates in the formation of the chain initiation complex, is generally believed to have a dual function: 1) It would ensure the availability of 30 S ribosomal subunits for initiation complex formation by promoting the dissociation of 70 S ribosomes [ 1,2]. This dissociation is usually ascribed to the binding of IF-3 to the 30 S sub-unit which would result in a shiftofequilibrium70S~========~3OSt 50 S towards the right [3,4]. 2) IF-3 would recognize some specific messenger starting signal on phage mRNA which is more complex than the initiator codon [5-71. Consistent with this second function is the finding by the group of Bosch [S] that the binding of ribosomes to phage MS2-RNA requires IF-3, whereas binding to synthetic messengers (containing the initiator codon ApUpG and a random base sequence) can occur in the absence of this factor. The isolation of two messenger-discriminating species of IF-3 is also in favor of different classes of mRNA being recognized by IF-3 [9]. It is therefore critical to know whether IF-3 is required or not in the translation of synthetic mRNAs. _ Several authys suggest that IF-3 is essential in the formation-of the initiation complex, not only with phage mRNA, but also with synthetic messenger [lo151. However, most of the experiments were performed with 70 S ribosomes, and the stimulating activity of IF-3 could be a consequence of its

Inhibition by thiostrepton of the IF‐2‐dependent ribosomal GTPase

During protein synthesis GTP hydrolysis occurs at the initiation and elongation steps [l] ; it has also been shown at the termination step in eukaryotic cells [2] . A GTP hydrolysis not coupled with protein synthesis (in the absence of mRNA and tRNA) can also be observed: whereas in the presence of either ribosome or any factor alone very little GTP hydrolysis can be observed, significant hydrolysis occurs in the presence of ribosomes and one of the factors involved in any one of the steps: initiation factor IF-2, or elongation factor EF-G [3] , or termination factor TF-R in eukaryotic cells [4-61. Some ribosomedependent GTP hydrolysis also occurs in thermophilic bacteria in the presence of EF-Tu [7] . The significance of these uncoupled GTP hydrolyses is not clear. Two groups of inhibitors of the factor and ribosome dependent GTP hydrolysis are known: the first, fusidic acid, prevents EF-G-dependent GTP hydrolysis by stabilizing the ternary complex: ribosome-EF-GGDP (which is formed after one round of EF-Gpromoted GTP hydrolysis), thus blocking any further GTP hydrolysis [8,9]. This antibiotic has no effect on the ribosomal GTPase promoted by IF-2 [S] , EF-T [7], or TF-R [2]. The second group includes thiostrepton and siomycin. These antibiotics inhibit EF-G and EF-T-dependent GTP hydrolysis by quite a different mechanism: they irreversibly inactivate the binding site for EF-G, thus preventing the formation of the ribo-

The Escherichia coli threonyl‐tRNA synthetase gene contains a split ribosomal binding site interrupted by a hairpin structure that is essential for autoregulation

The expression of the gene encoding Escherichia coli threonyl‐tRNA synthetase (ThrRS) is negatively autoregulated at the translational level. ThrRS binds to its own mRNA leader, which consists of four structural and functional domains: the Shine–Dalgarno (SD) sequence and the initiation codon region (domain 1); two upstream hairpins (domains 2 and 4) connected by a single‐stranded region (domain 3). Using a combination of in vivo and in vitro approaches, we show here that the ribosome binds to thrS mRNA at two non‐contiguous sites: region −12 to +16 comprising the SD sequence and the AUG codon and, unexpectedly, an upstream single‐stranded sequence in domain 3. These two regions are brought into close proximity by a 38‐nucleotide‐long hairpin structure (domain 2). This domain, although adjacent to the 5′ edge of the SD sequence, does not inhibit ribosome binding as long as the single‐stranded region of domain 3 is present. A stretch of unpaired nucleotides in domain 3, but not a specific sequence, is required for efficient translation. As the repressor and the ribosome bind to interspersed domains, the competition between ThrRS and ribosome for thrS mRNA binding can be explained by steric hindrance.

Escherichia coli phenylalanyl-tRNA synthetase operon is controlled by attenuation in vivo

The two subunits of phenylalanyl-tRNA synthetase are made from two adjacent, cotranscribed genes that constitute the pheS,T operon. Three different fusions between pheS,T and lac genes were constructed in order to study the regulation of the pheS,T operon in vivo. We show, using these fusions, that phenylalanyl-tRNA synthetase transcription is derepressed when the level of aminoacylated tRNAPhe is lowered by mutational alteration of the synthetase. The pheS,T operon is also derepressed in strains carrying a trpX mutation. The gene trpX codes for an enzyme that modifies both tRNATrp and tRNAPhe and a mutation in that gene causes derepression of the trp and pheA operons, both of which are controlled by attenuation. The in vivo features of the regulation of pheS,T expression described here in correlation with the DNA sequence and in vitro transcription results described in the accompanying paper by Fayat et al. indicate that phenylalanyl-tRNA synthetase is controlled by attenuation in a way analogous to several amino acid biosynthetic operons.

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.

Physiological effects of translation initiation factor IF3 and ribosomal protein L20 limitation in Escherichia coli.

To investigate the physiological roles of translation initiation factor IF3 and ribosomal protein L20 inEscherichia coli, theinfC, rpmI andrpIT genes encoding IF3, L35 and L20, respectively, were placed under the control oflac promoter/operator sequences. Thus, their expression is dependent upon the amount of inducer isopropyl thiogalactoside (IPTG) in the medium. Lysogenic strains were constructed with recombinant lambda phages that express eitherrpmI andrplT orinfC andrpmI in trans, thereby allowing depletion of only IF3 or L20 at low IPTG concentrations. At low IPTG concentrations in the IF3-limited strain, the cellular concentration of IF3, but not L20, decreases and the growth rate slows. Furthermore, ribosomes run off polysomes, indicating that IF3 functions during the initiation phase of protein synthesis in vivo. During slow growth, the ratio of RNA to protein increases rather than decreases as occurs with control strains, indicating that IF3 limitation disrupts feedback inhibition of rRNA synthesis. As IF3 levels drop, expression from an AUU-infC-lacZ fusion increases, whereas expression decreases from an AUG-infC-lacZ fusion, thereby confirming the model of autogenous regulation ofinfC. The effects of L20 limitation are similar; cells grown in low concentrations of IPTG exhibited a decrease in the rate of growth, a decrease in cellular L20 concentration, no change in IF3 concentration, and a small increase in the ratio of RNA to protein. In addition, a decrease in 50S subunits and the appearance of an aberrant ribosome peak at approximately 41–43S is seen. Previous studies have shown that the L20 protein negatively controls its own gene expression. Reduction of the cellular concentration of L20 derepresses the expression of anrplT-lacZ gene fusion, thus confirming autogenous regulation by L20.