Peptide bond formation and peptide release are catalyzed in the active site of the large subunit of the ribosome where universally conserved nucleotides surround the CCA ends of the peptidyl- and aminoacyl-tRNA substrates. Here, we describe the use of an affinity-tagging system for the purification of mutant ribosomes and analysis of four universally conserved nucleotides in the innermost layer of the active site: A2451, U2506, U2585, and A2602. While pre-steady-state kinetic analysis of the peptidyl transferase activity of the mutant ribosomes reveals substantially reduced rates of peptide bond formation using the minimal substrate puromycin, their rates of peptide bond formation are unaffected when the substrates are intact aminoacyl-tRNAs. These mutant ribosomes do, however, display substantial defects in peptide release. These results reveal a view of the catalytic center in which an inner shell of conserved nucleotides is pivotal for peptide release, while an outer shell is responsible for promoting peptide bond formation.

SUMMARY A crucial step in protein translation is the translocation of tRNAs through the ribosome. In the transition from one canonical site to the other, the tRNAs acquire intermediate configurations, so-called hybrid states. At this stage, the small subunits is rotated with respect to the large subunit, and the anticodon stem loops reside in the A and P sites of the small subunit, while the acceptor ends interact with the P and E sites of the large subunit. In this work, by means of cryo-EM and particle classification procedures, we visualize for the first time the hybrid state of both A/P and P/E tRNAs in an authentic factor-free ribosome complex during translocation. In addition, we show how the repositioning of the tRNAs goes hand in hand with the change in the interplay between S13, L1 stalk, L5, H68, H69 and H38 that is caused by the ratcheting of the small subunit.

Protein output from synonymous codons is thought to be equivalent if appropriate tRNAs are sufficiently abundant. Here we show that mRNAs encoding iterated lysine codons, AAA or AAG, differentially impact protein synthesis: insertion of iterated AAA codons into an ORF diminishes protein expression more than insertion of synonymous AAG codons. Kinetic studies in E. coli reveal that differential protein production results from pausing on consecutive AAA-lysines followed by ribosome sliding on homopolymeric A sequence. Translation in a cell-free expression system demonstrates that diminished output from AAA-codon-containing reporters results from premature translation termination on out of frame stop codons following ribosome sliding. In eukaryotes, these premature termination events target the mRNAs for Nonsense-Mediated-Decay (NMD). The finding that ribosomes slide on homopolymeric A sequences explains bioinformatic analyses indicating that consecutive AAA codons are under-represented in gene-coding sequences. Ribosome ‘sliding’ represents an unexpected type of ribosome movement possible during translation.DOI: http://dx.doi.org/10.7554/eLife.05534.001

The GTPase elongation factor (EF)-G is responsible for promoting the translocation of the messenger RNA-transfer RNA complex on the ribosome, thus opening up the A site for the next aminoacyltRNA. Chemical modification and cryo-EM studies have indicated that tRNAs can bind the ribosome in an alternative 'hybrid' state after peptidyl transfer and before translocation, though the relevance of this state during translation elongation has been a subject of debate. Here, using pre-steady-state kinetic approaches and mutant analysis, we show that translocation by EF-G is most efficient when tRNAs are bound in a hybrid state, supporting the argument that this state is an authentic intermediate during translation.Translation elongation is the multistep process performed by the ribosome to sequentially add mRNA-encoded amino acids to the growing polypeptide chain. There are three iterated steps performed by the ribosome during the elongation cycle: (i) a tRNA-selection step in which the ribosome and elongation factor (EF)-Tu select the next aminoacyl-tRNA to enter the cycle; (ii) peptide-bond formation catalyzed in the active site of the large ribosomal subunit; and (iii) translocation of the mRNA-tRNA complex through the subunit interface region, facilitated by EF-G, with associated GTP hydrolysis. The tRNA substrates are at the heart of each of these steps, where they have key functional roles 1-3 .Early studies on the ribosome identified binding sites for two tRNA substrates, the A site for the aminoacyl-tRNA and the P site for the peptidyl-tRNA. Further biochemistry eventually identified a third site, the E or exit site, where deacylated tRNAs bind after loss of the peptidyl moiety and before release into solution 4 . An understanding of the order and timing with which tRNA substrates occupy these three sites (on both the large and small subunits) during the elongation cycle is central to a detailed molecular view of the process of translation.The hybrid state of tRNA binding on the ribosome was first proposed in the 1960s as an elegant way to understand how controlled tRNA movement through the ribosome might be accomplished 5 . The basic feature of such a hybrid state of binding is that tRNAs would move independently with respect to the two subunits of the ribosome during the steps of the translation cycle. Fluorescence studies provided early biochemical clues that such a state of tRNA binding might be populated during translation 6 , and subsequent chemical-modification analysis provided clear and detailed experimental support for the hybrid state 7 . These studies Correspondence should be addressed to R.G. (ragreen@jhmi.edu).. Note: Supplementary information is available on the Nature Structural & Molecular Biology website. COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.Published online at http://www.nature.com/nsmb/ Reprints and permissions information is available online at http://www.nature.com/reprints/index.html NIH Public Access Fig. 1a). Thus, 'hybr...

Ribosomal variants carrying mutations in active site nucleotides are severely compromised in their ability to catalyze peptide bond formation (PT) with minimal aminoacyl tRNA substrates such as puromycin. However, catalysis of PT by these same ribosomes with intact aminoacyl tRNA substrates is uncompromised. These data suggest that these active site nucleotides play an important role in the positioning of minimal aminoacyl tRNA substrates but are not essential for catalysis per se when aminoacyl tRNAs are positioned by more remote interactions with the ribosome. Previously reported biochemical studies and atomic resolution X-ray structures identified a direct Watson-Crick interaction between C75 of the A-site substrate and G2553 of the 23S rRNA. Here we show that the addition of this single cytidine residue (the C75 equivalent) to puromycin is sufficient to suppress the deficiencies of active site ribosomal variants, thus restoring ''tRNA-like'' behavior to this minimal substrate. Studies of the binding parameters and the pH-dependence of catalysis with this minimal substrate indicate that the interaction between C75 and the ribosomal A loop is an essential feature for robust catalysis and further suggest that the observed effects of C75 on peptidyl transfer activity reflect previously reported conformational rearrangements in this active site.

Translocation of the mRNA:tRNA complex through the ribosome is promoted by elongation factor G (EF-G) during the translation cycle. Previous studies established that modification of ribosomal proteins with thiol-specific reagents promotes this event in the absence of EF-G. Here we identify two small subunit interface proteins S12 and S13 that are essential for maintenance of a pretranslocation state. Omission of these proteins using in vitro reconstitution procedures yields ribosomal particles that translate in the absence of enzymatic factors. Conversely, replacement of cysteine residues in these two proteins yields ribosomal particles that are refractive to stimulation with thiol-modifying reagents. These data support a model where S12 and S13 function as control elements for the more ancient rRNA- and tRNA-driven movements of translocation.

Accurate discrimination between cognate and near-cognate aminoacyl-tRNAs during translation relies on the specific acceleration of forward rate constants for cognate tRNAs. Such specific rate enhancement correlates with conformational changes in the tRNA and small ribosomal subunit that depend on an RNA-specific type of interaction, the A-minor motif, between universally conserved 16S ribosomal RNA nucleotides and the cognate codon-anticodon helix. We show that perturbations of these two components of the A-minor motif, the conserved rRNA bases and the codon-anticodon helix, result in distinct outcomes. Although both cause decreases in the rates of tRNA selection that are rescued by aminoglycoside antibiotics, only disruption of the codon-anticodon helix is overcome by a miscoding tRNA variant. On this basis, we propose that two independent molecular requirements must be met to allow tRNAs to proceed through the selection pathway, providing a mechanism for exquisite control of fidelity during this step in gene expression.

Small interfering RNAs (siRNAs) and microRNAs (miRNAs) bind to Argonaute family proteins to form a related set of effector complexes that play diverse roles in post-transcriptional gene regulation throughout the eukaryotic lineage. Here, sequence and structural analysis of the MID domain of the Argonaute proteins identified similarities with a family of allosterically regulated bacterial ligandbinding domains. In vitro and in vivo approaches were used to show that certain Argonaute proteins (those involved in translational repression) have conserved this functional allostery between two distinct sites, one involved in binding miRNA:target duplex and the other in binding the 5' cap feature (m 7 GpppG) of eukaryotic mRNAs. This allostery provides an explanation for how miRNA-bound effector complexes may avoid indiscriminate repressive action (mediated through binding interactions with the cap) prior to full target recognition. KeywordsArgonaute; miRNA; miRNP; m 7 GpppG cap; allostery Small interfering RNAs (siRNAs) and microRNAs (miRNAs) belong to an increasingly broad class of small, non-coding RNA molecules found in diverse organisms 1 . These two groups are broadly distinguished by their biogenesis pathways and their differential loading into distinct Argonaute complexes. siRNAs are generated in the cytoplasm and are loaded into an Argonaute-containing RNA-induced silencing complex (siRISC) to cleave targets with perfect complementarity. miRNAs are transcribed in the nucleus from a specific gene and are ultimately loaded into Argonaute-containing miRNPs (miRISC) that limit the expression of distinct mRNA targets with imperfect complementarity. And, though there are clearly shared molecular features for these two processes (e.g. an Argonaute protein and a small RNA), there are also distinct molecular features and players important both for small RNA loading and target recognition 2 . The Argonaute proteins are composed of several distinct domains with partially understood functions: an N-terminal domain, a PAZ domain that contains the binding site for the 3' end of the small RNA, a MID domain that contains the binding site for the 5' end of the small RNA (which will be the focus of discussion in this manuscript), and the PIWI domain that contains the catalytic center for the cleavage reaction that occurs during RNA interference (Fig. 1a).A number of molecular mechanisms have been proposed to account for the observed posttranscriptional control of miRNA-targeted genes including inhibition of translation initiation or elongation and the promotion of mRNA decay (reviewed in ref. 3). One previous studyCorrespondence should be addressed to R.G. (ragreen@jhmi.edu).. * These authors contributed equally to this work. Author InformationThe authors declare no competing financial interests. NIH Public Access Author ManuscriptNat Struct Mol Biol. Author manuscript; available in PMC 2010 March 8. RESULTS Argonaute MID domain analysis reveals functional groupingsThe mechanistic questions that arose from the studies b...