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Bacterial translation termination is triggered when a stop codon arrives at the ribosomal A site. Stop codons are recognized by class I release factors (RF1 and RF2 in Escherichia coli), which bind to the ribosome and catalyze the release of the newly synthesized protein. Crystal structures showed that RF1 and RF2 are in an open conformation when bound to the ribosome but are in a closed conformation when not bound to the ribosome. It is not clear whether only the open form of RF1 and RF2 binds to the ribosome. Alternatively, the closed form of RF1 and RF2 may bind to the ribosome and undergo a conformational change to the open state upon binding. We used transition metal ion fluorescence resonance energy transfer experiments to monitor precisely the conformation of RF1 in the absence and presence of the ribosome. Our results indicate that RF1 undergoes a large conformational change from a closed to an open form upon binding to the ribosome. Our results are consistent with the mechanism, in which high termination fidelity is achieved by linking stop codon recognition by RF1 to the change in conformation from closed to open state.

Bacterial translation initiation factor 3 (IF3) is involved in the fidelity of translation initiation at several levels, including start-codon discrimination, mRNA translation, and initiator-tRNA selection. The IF3 C-terminal domain (CTD) is required for binding to the 30S ribosomal subunit. N-terminal domain (NTD) function is less certain, but likely contributes to initiation fidelity. Point mutations in either domain can decrease initiation fidelity, but C-terminal domain mutations may be indirect. Here, the Y75N substitution mutation in the NTD is examined in vitro and in vivo. IF3(Y75N) protein binds 30S subunits normally, but is defective in start-codon discrimination, inhibition of initiation on leaderless mRNA, and initiator-tRNA selection, thereby establishing a direct role for the IF3 NTD in these initiation processes. A model illustrating how IF3 modulates an inherent function of the 30S subunit is discussed.

Bacterial translation initiation factor IF2 was localized on the ribosome by rRNA cleavage using free Cu(II):1,10-orthophenanthroline. The results indicated proximity of IF2 to helix 89, to the sarcin-ricin loop and to helices 43 and 44, which constitute the "L11/thiostrepton" stem-loops of 23S rRNA. These findings prompted an investigation of the L11 contribution to IF2 activity and a re-examination of the controversial issue of the effect on IF2 functions of thiostrepton, a peptide antibiotic known primarily as a powerful inhibitor of translocation. Ribosomes lacking L11 were found to have wild-type capacity to bind IF2 but a strongly reduced ability to elicit its GTPase activity. We found that thiostrepton caused a faster recycling of this factor on and off the 70S ribosomes and 50S subunits, which in turn resulted in an increased rate of the multiple turnover IF2-dependent GTPase. Although thiostrepton did not inhibit the P-site binding of fMet-tRNA, the A-site binding of the EF-Tu-GTP-Phe-tRNA or the activity of the ribosomal peptidyl transferase center (as measured by the formation of fMet-puromycin), it severely inhibited IF2-dependent initiation dipeptide formation. This inhibition can probably be traced back to a thiostrepton-induced distortion of the ribosomal-binding site of IF2, which leads to a non-productive interaction between the ribosome and the aminoacyl-tRNA substrates of the peptidyl transferase reaction. Overall, our data indicate that the translation initiation function of IF2 is as sensitive as the translocation function of EF-G to thiostrepton inhibition.

Heterotrimeric aIF2αβγ (archaeal homologue of the eukaryotic translation initiation factor 2) in its GTP-bound form delivers Met-tRNA i Met to the small ribosomal subunit. It is known that the heterodimer containing the GTP-bound γ subunit and domain 3 of the α subunit of aIF2 is required for the formation of a stable complex with Met-tRNA i . Here, the crystal structure of an incomplete ternary complex including aIF2αD3γ ⋅ GDPNP ⋅ Met-tRNA f Met has been solved at 3.2 A resolution. This structure is in good agreement with biochemical and hydroxyl radical probing data. The analysis of the complex shows that despite the structural similarity of aIF2γ and the bacterial translation elongation factor EF-Tu, their modes of tRNA binding are very different. Remarkably, the recently published 5.0-A-resolution structure of almost the same ternary initiation complex differs dramatically from the structure presented. Reasons for this discrepancy are discussed.

The mechanisms for de novo protein folding differ significantly between bacteria and eukaryotes, as evidenced by the often observed poor yields of native eukaryotic proteins upon recombinant production in bacterial systems. Polypeptide synthesis rates are faster in bacteria than in eukaryotes, but the effects of general variations in translation rates on protein folding efficiency have remained largely unexplored. By employing Escherichia coli cells with mutant ribosomes whose translation speed can be modulated, we show here that reducing polypeptide elongation rates leads to enhanced folding of diverse proteins of eukaryotic origin. These results suggest that in eukaryotes, protein folding necessitates slow translation rates. In contrast, folding in bacteria appears to be uncoupled from protein synthesis, explaining our findings that a generalized reduction in translation speed does not adversely impact the folding of the endogenous bacterial proteome. Utilization of this strategy has allowed the production of a native eukaryotic multidomain protein that has been previously unattainable in bacterial systems and may constitute a general alternative to the production of aggregation-prone recombinant proteins.

We reported elsewhere (Varenne et al., 1982) that, during synthesis of a number of colicins in Escherichia coli, intermediate nascent chains of discrete sizes accumulated, suggesting a variable rate of translation. In this paper, a detailed analysis provides arguments that this phenomenon, at least for the proteins under study, is not related to aspects of messenger RNA such as secondary structure. It is linked to the difference in transfer RNA availability for the various codons. Experimental analysis of translation of other proteins in E. coli confirms that the main origin for the discontinuous translation in the polypeptide elongation cycle is the following. For a given codon, the stochastic search of the cognate ternary complex (aminoacyl-tRNA-EF-Tu-GTP) is the rate-limiting step in the elongation cycle: transpeptidation and translocation steps are much faster. The degree of slackening in ribosome movement is almost proportional to the inverse of tRNA concentrations. The verification of this model and its possible physiological significance are discussed.