Summary Translational control is frequently exerted at the stage of mRNA recruitment to the initiating ribosome. We have reconstituted mRNA recruitment to the 43S preinitiation complex (PIC) using purified S. cerevisiae components. We show that eIF3 and the eIF4 factors not only stabilize binding of mRNA to the PIC, they also dramatically increase the rate of recruitment. Although capped mRNAs require eIF3 and the eIF4 factors for efficient recruitment to the PIC, uncapped mRNAs can be recruited in the presence of eIF3 alone. The cap strongly inhibits this alternative recruitment pathway, imposing a requirement for the eIF4 factors for rapid and stable binding of natural mRNA. Our data suggest that the 5′-cap serves as both a positive and negative element in mRNA recruitment, promoting initiation in the presence of the canonical group of mRNA handling factors while preventing binding to the ribosome via an aberrant, alternative pathway requiring only eIF3.

Eukaryotic translation initiation factor (eIF)4B stimulates recruitment of mRNA to the 43S ribosomal pre-initiation complex (PIC). Yeast eIF4B (yeIF4B), shown previously to bind single-stranded (ss) RNA, consists of an N-terminal domain (NTD), predicted to be unstructured in solution; an RNA-recognition motif (RRM); an unusual domain comprised of seven imperfect repeats of 26 amino acids; and a C-terminal domain. Although the mechanism of yeIF4B action has remained obscure, most models have suggested central roles for its RRM and ssRNA-binding activity. We have dissected the functions of yeIF4B's domains and show that the RRM and its ssRNA-binding activity are dispensable in vitro and in vivo. Instead, our data indicate that the 7-repeats and NTD are the most critical domains, which mediate binding of yeIF4B to the head of the 40S ribosomal subunit via interaction with Rps20. This interaction induces structural changes in the ribosome's mRNA entry channel that could facilitate mRNA loading. We also show that yeIF4B strongly promotes productive interaction of eIF4A with the 43S•mRNA PIC in a manner required for efficient mRNA recruitment.

A widely held view is that directional movement of tRNA in the ribosome is determined by an intrinsic mechanism and driven thermodynamically by transpeptidation. Here, we show that, in certain ribosomal complexes, the pretranslocation (PRE) state is thermodynamically favored over the posttranslocation (POST) state. Spontaneous and efficient conversion from the POST to PRE state is observed when EF-G is depleted from ribosomes in the POST state or when tRNA is added to the E site of ribosomes containing P-site tRNA. In the latter assay, the rate of tRNA movement is increased by streptomycin and neomycin, decreased by tetracycline, and not affected by the acylation state of the tRNA. In one case, we provide evidence that complex conversion occurs by reverse translocation (i.e., direct movement of the tRNAs from the E and P sites to the P and A sites, respectively). These findings have important implications for the energetics of translocation.

During translation, tRNAs must move rapidly to their adjacent sites in the ribosome while maintaining precise pairing with mRNA. This movement (translocation) occurs in a stepwise manner with hybrid-state intermediates, but it is unclear how these hybrid states relate to kinetically defined events of translocation. Here we analyze mutations at position 2394 of 23S rRNA in a pre-steadystate kinetic analysis of translocation. These mutations target the 50S E site and are predicted to inhibit P/E state formation. Each mutation decreases growth rate, the maximal rate of translocation (k trans), and the apparent affinity of EF-G for the pretranslocation complex (i.e., increases K 1/2). The magnitude of these defects follows the trend A > G > U. Because the C2394A mutation did not decrease the rate of single-turnover GTP hydrolysis, the >20-fold increase in K 1/2 conferred by C2394A can be attributed to neither the initial binding of EF-G nor the subsequent GTP hydrolysis step. We propose that C2394A inhibits a later step, P/E state formation, to confer its effects on translocation. Replacement of the peptidyl group with an aminoacyl group, which is predicted to inhibit A/P state formation, decreases k trans without increasing K1/2. These data suggest that movements of tRNA into the P/E and A/P sites are separable events. This mutational study allows tRNA movements with respect to both subunits to be integrated into a kinetic model for translocation.elongation factor G ͉ translation ͉ exit site ͉ GTPase M ovement of tRNAs through the ribosome is believed to occur in a stepwise manner (1). Chemical protection experiments showed that, after peptidyl transfer, the acceptor ends of the tRNAs can move spontaneously with respect to the 50S subunit to form the hybrid state. In the hybrid state, the deacylated tRNA occupies the 30S P site and 50S E site (P/E site) while the peptidyl-tRNA occupies the 30S A site and 50S P site (A/P site). It was proposed that hybrid state formation precedes codon-anticodon movement within the 30S subunit, and only the latter event requires catalysis by elongation factor G (EF-G). Support for the hybrid-state model came from Förster resonance energy transfer (FRET) experiments in which an Ϸ20-Å movement of the 5Ј end of the newly deacylated tRNA toward ribosomal protein L1 was inferred after peptide bond formation, whereas the position of the peptidyl group changed little (2). Because L1 lies near the 50S E site, these data are consistent with movement of tRNA into the P/E site. Further evidence came from single-molecule FRET studies that monitored the distance between probes attached to the elbows of tRNAs in the A and P sites. Fluctuations between two distinct configurations were observed that were structurally consistent with the classical and hybrid states (3). More recently, a third state of the pretranslocation (PRE) complex was observed by single-molecule FRET, consistent with one tRNA bound to the P/E site and the other bound to the A/A site (4). Experiments that monitored mRNA in ribos...

eIF4G is the scaffold subunit of the eIF4F complex, whose binding domains for eIF4E and poly(A)-binding protein (PABP) are thought to enhance formation of activated eIF4F K mRNA K PABP complexes competent to recruit 43S pre-initiation complexes. We found that the RNA-binding region (RNA1) in the N-terminal domain (NTD) of yeast eIF4G1 can functionally substitute for the PABP-binding segment to rescue the function of an eIF4G1-459 mutant impaired for eIF4E binding. Assaying RNA-dependent PABP-eIF4G association in cell extracts suggests that RNA1, the PABP-binding domain, and two conserved elements (Box1 and Box2) between these segments have overlapping functions in forming native eIF4GKmRNAK PABP complexes. In vitro experiments confirm the role of RNA1 in stabilizing eIF4G-mRNA association, and further indicate that RNA1 and Box1 promote PABP binding, in addition to RNA binding, by the eIF4G1 NTD. Our findings indicate that PABP-eIF4G association is only one of several interactions that stabilize eIF4FKmRNA complexes, and emphasize that closed-loop mRNP formation via PABP-eIF4G interaction is non-essential in vivo. Interestingly, two other RNA-binding regions in eIF4G1 have critical functions downstream of eIF4F K mRNA assembly.

In the cell, the activity of tRNA is governed by its acylation state. Interactions with the ribosome, translation factors, and regulatory elements are strongly influenced by the acyl group, and presumably other cellular components that interact with tRNA also use the acyl group as a specificity determinant. Thus, those using biochemical approaches to study any aspect of tRNA biology should be familiar with effective methods to prepare and evaluate acylated tRNA reagents. Here, methods to prepare aminoacyl-tRNA, N-acetyl-aminoacyl-tRNA, and fMet-tRNA(fMet) and to assess their homogeneity are described. Using these methods, acylated tRNAs of high homogeneity can be reliably obtained.

Protein synthesis occurs in ribosomes, the targets of numerous antibiotics. How these large and complex machines read and move along mRNA have proven to be challenging questions. In this Review, we focus on translocation, the last step of the elongation cycle in which movement of tRNA and mRNA is catalyzed by elongation factor G. Translocation entails large-scale movements of the tRNAs and conformational changes in the ribosome that require numerous tertiary contacts to be disrupted and reformed. We highlight recent progress toward elucidating the molecular basis of translocation and how various antibiotics influence tRNA-mRNA movement.In all cells, proteins are synthesized by ribosomes, megadalton RNA-protein machines that use aminoacyl-tRNA (aa-tRNA) molecules to translate messenger RNA (mRNA). Each ribosome is composed of a large and small subunit. The Escherichia coli large (50S) subunit consists of 2 RNA molecules (23S, 2904 nt; 5S, 120 nt) and 34 proteins (L1-L7 and L9-L36), whereas the small (30S) subunit consists of 1 RNA molecule (16S, 1542 nt) and 21 proteins (S1-S21). During translation, incorporation of each amino acid into the nascent polypeptide chain involves three sequential steps: decoding, peptidyl transfer, and translocation ( Figure 1). Decoding is facilitated by elongation factor Tu (EF-Tu), which delivers aa-tRNA to the A (aminoacyl) site as part of a ternary complex with GTP (1,2). When codon recognition occurs in the 30S A site, EF-Tu is activated to hydrolyze GTP, which promotes release of the acceptor end of aa-tRNA and its movement into the 50S A site. Once aa-tRNA is in the A site of both subunits, the ribosome catalyzes transfer of the peptidyl group of P-site tRNA to A-site aa-tRNA. This leaves a complex containing peptidyl-tRNA in the A site and deacylated tRNA in the P site, termed the pretranslocation (PRE) complex. Translocation of the tRNAs to their adjacent sites is then catalyzed by elongation factor G (EF-G), which hydrolyzes GTP in the process. Translocation is believed to occur in a stepwise manner. First, the tRNA acceptor stems move within the 50S subunit to form the hybrid-state complex, where peptidyl-tRNA and deacylated tRNA occupy the A/ P and P/E sites, respectively. Then, the codon-anticodon helices move within the 30S subunit to form the post-translocation (POST) complex, in which peptidyl-tRNA and deacylated tRNA occupy the P and E sites of both subunits (P/P and E/E sites). This leaves the A site vacant, ready for the next round of elongation.

In contrast to elongation factor EF-Tu, which delivers aminoacyl-tRNAs to the ribosomal A-site, eukaryotic initiation factor eIF2 binds initiator Met-tRNAiMet to the P-site of the 40S ribosomal subunit. We used directed hydroxyl radical probing experiments to map the binding of Saccharomyces cerevisiae eIF2 on the ribosome and on Met-tRNAiMet. Our results identify a key binding-interface between domain III of eIF2γ and 18S rRNA helix h44 on the 40S subunit. Moreover, we showed that eIF2γ primarily contacts the acceptor stem of Met-tRNAiMet. Whereas the analogous domain III of EF-Tu contacts the T-stem of tRNAs, biochemical analyses demonstrated that eIF2γ domain III is important for ribosome, but not Met-tRNAiMet, binding. Thus despite their structural similarity, eIF2 and EF-Tu bind tRNAs in substantially different manners, and we propose that the tRNA-binding domain III of EF-Tu has acquired a new ribosome-binding function in eIF2γ.