The complex of tmRNA-SmpB and EF-G on translocating ribosomes.

Bacterial ribosomes stalled at the 3' end of malfunctioning messenger RNAs can be rescued by transfer-messenger RNA (tmRNA)-mediated trans-translation. The SmpB protein forms a complex with the tmRNA, and the transfer-RNA-like domain (TLD) of the tmRNA then enters the A site of the ribosome. Subsequently, the TLD-SmpB module is translocated to the P site, a process that is facilitated by the elongation factor EF-G, and translation is switched to the mRNA-like domain (MLD) of the tmRNA. Accurate loading of the MLD into the mRNA path is an unusual initiation mechanism. Despite various snapshots of different ribosome-tmRNA complexes at low to intermediate resolution, it is unclear how the large, highly structured tmRNA is translocated and how the MLD is loaded. Here we present a cryo-electron microscopy reconstruction of a fusidic-acid-stalled ribosomal 70S-tmRNA-SmpB-EF-G complex (carrying both of the large ligands, that is, EF-G and tmRNA) at 8.3 Å resolution. This post-translocational intermediate (TI(POST)) presents the TLD-SmpB module in an intrasubunit ap/P hybrid site and a tRNA(fMet) in an intrasubunit pe/E hybrid site. Conformational changes in the ribosome and tmRNA occur in the intersubunit space and on the solvent side. The key underlying event is a unique extra-large swivel movement of the 30S head, which is crucial for both tmRNA-SmpB translocation and MLD loading, thereby coupling translocation to MLD loading. This mechanism exemplifies the versatile, dynamic nature of the ribosome, and it shows that the conformational modes of the ribosome that normally drive canonical translation can also be used in a modified form to facilitate more complex tasks in specialized non-canonical pathways.

New features of the ribosome and ribosomal inhibitors: non-enzymatic recycling, misreading and back-translocation.

We describe the optimization of a poly(Phe) synthesis system, the conditions of which have been applied for efficient translation of heteropolymeric mRNAs. Here we identify two parameters that are essential to obtain translation at efficiency and accuracy levels equivalent to those in vivo, viz., the fine-tuning of the energy-rich components with an acetyl-phosphate substrate for energy regeneration, as well as the ionic conditions. Applying this system revealed a number of new features: (i) 70S ribosomes are able to recycle within 300 s in a non-enzymatic fashion in the absence of tmRNA. This observation might explain the fact that a knockout of the tmRNA gene ssrA is not lethal for Escherichia coli cells in contrast to other bacterial strains, such as Bacillus subtilis. (ii) The high efficiency of the system was exploited to analyze the misincorporation of various amino acids (resolution limit=1:15,000). No misreading was observed at the middle codon position and only marginal effects were observed at the first one (even when misreading was artificially stimulated 20- to 30-fold), yielding an improved definition of the near-cognate and non-cognate aminoacyl-tRNAs. (iii) Aminoglycosides increase Phe and Lys incorporation about 2-fold in the presence of poly(U) or poly(UUC) and poly(A), respectively, and induce a back-translocation (except hygromycin B) exclusively in the absence of EF-G*GTP, as do the non-related drugs viomycin and edeine.

Dissecting the ribosomal inhibition mechanisms of edeine and pactamycin: the universally conserved residues G693 and C795 regulate P-site RNA binding.

The crystal structures of the universal translation-initiation inhibitors edeine and pactamycin bound to ribosomal 30S subunit have revealed that edeine induces base pairing of G693:C795, residues that constitute the pactamycin binding site. Here, we show that base pair formation by addition of edeine inhibits tRNA binding to the P site by preventing codon-anticodon interaction and that addition of pactamycin, which rebreaks the base pair, can relieve this inhibition. In addition, edeine induces translational misreading in the A site, at levels comparable to those induced by the classic misreading antibiotic streptomycin. Binding of pactamycin between residues G693 and C795 strongly inhibits translocation with a surprising tRNA specificity but has no effect on translation initiation, suggesting that reclassification of this antibiotic is necessary. Collectively, these results suggest that the universally conserved G693:C795 residues regulate tRNA binding at the P site of the ribosome and influence translocation efficiency.

Protein synthesis at atomic resolution: mechanistics of translation in the light of highly resolved structures for the ribosome.

Our understanding of the process of translation has progressed rapidly since the availability of highly resolved structures for the ribosome. A wealth of information has emerged in terms of both RNA and protein structure and the interplay between them. This has prompted us to revisit the astonishing "treasure trove" of functional data regarding the ribosome that has accumulated over the past decades. Here we try a systematic synopsis of these ribosomal functions in light of the cryo-electron microscopic structures (resolution >7 A) and the atomic x-ray structures (>2.4 A) of the ribosome.

How does tmRNA move through the ribosome?

To test the structure of tmRNA in solution, cross-linking experiments were performed which showed two sets of cross-links in two different domains of tmRNA. Site-directed mutagenesis was used to search for tmRNA nucleotide bases that might form a functional analogue of a codon-anticodon duplex to be recognized by the ribosomal A-site. We demonstrate that nucleotide residues U85 and A86 from tmRNA are significant for tmRNA function and propose that they are involved in formation of a tmRNA element playing a central role in A-site recognition. These data are discussed in the frame of a hypothetical model that suggests a general scheme for the interaction of tmRNA with the ribosome and explains how it moves through the ribosome.