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.

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.

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.

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In order to identify the amino acid residues necessary for the selective recognition of the mRNA cap structure by human eukaryotic initiation factor-4E (eIF-4E), which plays a central role in the first step of mRNA translation, we prepared recombinant wild-type and fourteen mutant forms and compared their cap-binding abilities by affinity chromatography. By the direct expression of a synthetic gene encoding human eIF-4E as the soluble form in Escherichia coli and the application on a 7-methylguanosine-5'-triphosphate-Sepharose 4B cap affinity column, pure recombinant eIF-4E was prepared; the optimum pH for the binding of the mRNA cap was 7.5. Among the amino acid residues conserved among various eIF-4E species, each of 14 functional residues was replaced with a nonpolar amino acid (alanine or leucine). All mutant eIF-4E genes, which were constructed by site-directed niutagenesis, were expressed in the same way as the wild type, and their cap-binding abilities were compared with that of the wild type. Consequently, all eight tryptophan residues, Glu103, and two histidine residues at positions 37 and 200 in human recombinant eIF-4E were suggested to be important for the recognition of the mRNA cap structure through direct interaction and/or indirect contributions. Indirect contributions included the construction of the overall protein structure, especially the cap-binding pocket.Keywords: human eIF-4E ; m'G(5')ppp ; mRNA cap structure ; site-directed mutagenesis ; cap binding.Many eukaryotic mRNAs have a common cap structure [m7G(5')ppp(5')N, where N is any nucleotide] at the 5'-terminal portion and its structure plays important roles in stabilizing the mRNA structure and facilitating mRNA binding to ribosomes during initiation [l-31. To allow for the efficient translation of mRNA, however, an interaction is required between the cap structure and eukaryotic initiation factor (eIF)-4 polypeptides consisting of eIF-4A, eIF-4B, eIF-4E, and eIF-4y 143. eIF-4E, which corresponds to the smallest subunit of eIF-4F, has been shown to bind specifically to the mRNA cap structure and has an important function in the first step of protein synthesis; it appears to play a key role in the regulation of translation via phosphorylation [5 -71. Furthermore, recent studies have indicated that eIF-4E participates in the regulation of translation through the interaction with 4E-BP [8].The amino acid sequences of eIF-4Es from yeast [9], human [lo], mouse [ I l l , rabbit 1121, wheat germ [13], and Drosophila [14] have already been reported. Human eIF-4E is a polypeptide of about 25 kDa that contains eight tryptophan residues and its amino acid residues have been remarkably conserved in number and position during evolution among human, mouse, yeast, rabbit, wheat, and Drosophila, which probably reflects the importance for the recognition of the mRNA cap structure [15]. From fluorescence studies at various pH values, however, histidineCorrespondt.nce to T. Ishida, Department of Physical Chemistry, Osaka University of Pharmaceutical Scie...