Jacques Dondon

Light-scattering studies showing the effect of initiation factors on the reversible dissociation of Escherichia coli ribosomes.

Abstract Light scattering was used to determine the effects of the bacterial initiation factors on the 70 S ribosome subunit equilibrium: 70 S ⇌ k 2 k 1 30 S + 50 s. The equilibrium proportion of associated ribosomes was studied as a function of IF-3, IF-1 and IF-2 § concentrations, and the rate constants κ1 and κ2 were determined in the presence of IF-3 and IF-1. Our results strongly support the view that IF-3-induced dissociation of the 70 S couple results from its binding to the free 30 S subunit, thus shifting the equilibrium toward dissociation. IF-3 causes κ2 to decrease drastically, while κ1 is unaffected; moreover, equilibrium values obtained with A-type ribosomes (tight couples) are consistent with binding of IF-3 to the 30 S subunit (association constant K3 = 2·5 × 107 to 4·0 × 107 m −1) and with its exclusion from the 70 S ribosome. No significant variation of K3 was found, either with temperature (25 to 37 °C) or with Mg2+ concentration (2·2 to 5 m m ). IF-1 was found to aid IF-3-induced dissociation by increasing the rate constants κ1 and κ2. IF-1 alone showed a limited dissociating activity, interpreted as due to its stronger binding to the 30 S than to the 70 S particle. Accordingly, κ1 has a stronger dependence on IF-1 concentration than κ2. The affinity of IF-3 for the 30 S subunit was observed not to vary significantly on addition of IF-1, when both factors were used in nearly stoichiometric amounts (not more than threefold excess over the ribosomes). When, on the contrary, IF-1 and IF-3 were present in large excess, dissociation was more efficient than expected from simple one to one binding of the factors to the ribosome. Streptomycin slowed the rate of dissociation by IF-3 and IF-1, but did not entirely suppress the effect of the factors. IF-2 was found to cause association of the ribosomal subunits; the effect is stronger and more specific in the presence of GMPPCP or GTP than in their absence. In the latter case, increase of the light-scattering signal probably involves some aggregation. In. the presence of GMPPCP, data are consistent with the following binding constants of IF-2: K2 m −1 to the 30 S, and K2′ = 1 × 107 to 1·5 × 107 m −1 to the 70 S ribosomes.

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-

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.

Photosensitized cross-linking of IF-3 to Escherichia coli 30 S subunits

IF-3 is currently believed to have a dual function in protein synthesis. Initiation pro- ceeds via dissociation of 70 S ribosomes into 30 S and 50 S subunits, and by binding tightly to the 30 S subunit, IF-3 favors this process. In addition, IF-3 appears to direct binding of 30 S subunits to start signals in messenger RNA [l] . A structurally detailed mechanism accounting for this dual functionality has been proposed [2], an important test for which would be the localization of the IF-3 binding site on the 30 S particle. Dye-photosensitized cross-linking of nucleic acids to proteins has been reported previously [3,4]. We here present results describing the successful application of a photosensitization procedure for inducing cross-links between IF-3 and both 30 S proteins and 16 S RNA. 2. Materials and methods Purified IF-3 was prepared as described previously [5]. IF-3 was labeled via reductive methylation, using [14C] formaldehyde [New England Nuclear, 44 Ci/mol] and sodium borohydride [6]. Typically, labeling of 3000-5000 cpm/pg was achieved. Microprotein determinations were accomplished using the method of Schaffner and Weissmann [7]. Labeled protein was freed from excess formaldehyde by Sephadex G-75 filtration. In accord with results previously reported [8 1, methylation led to no loss in IF-3 activity as measured by stimulation of fMet--tRNAmef

IF-3 crosslinking to Escherichia coli ribosomal 30 S subunits by three different light-dependent procedures: Identification of 30 S proteins crosslinked to IF-3—Utilization of a new two-stage crosslinking reagent, p-nitrobenzylmaleimide

Abstract We here report the results of using three light-dependent procedures for crosslinking IF-3 to 30 S proteins within an IF-3·30 S complex. In the first procedure, employing FMN as a photosensitizer, protein S12 is found to be the only major crosslinked protein. In the second procedure, IF-3 is first reacted with the new two-stage crosslinking reagent, p -nitrobenzylmaleimide (PNBM), and the PNBM—IF-3·30 S complex is irradiated. The major crosslinked proteins are S3 > S2, S12, S18. Small amounts of crosslinked S11 and S21 are also found. In the third procedure, the IF-3·30 S complex is reacted with PNBM and then irradiated. The major crosslinked proteins are S12 > S3 > S11 and small amounts of crosslinked S1, S13, and S21 are also found. These results are compared with results obtained by others using different crosslinking procedures and are used to discuss the Lake and Kahan model ( J. A. Lake and L. Kahan, 1975, J. Mol. Biol. , 99 , 631–644 , and J. A. Lake, 1978, in Advanced Techniques in Biological Electron Microscopy II, Koehler, J. K., ed., pp. 173–211, Springer-Verlag, Berlin ) for IF-3 binding to 30 S subunits.

Translational control inE. coli: The case of threonyl-tRNA synthetase

Genetic studies have shown that expression of theE. 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 50bp upstream of the translational initiation codon in thethrS gene, is directly involved in that control. The conformation of anin vitro RNA fragment extending over thethrS 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 constitutent 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.