Observation of Intersubunit Movement of the Ribosome in Solution Using FRET

112

In cap-dependent translation initiation, the open reading frame (ORF) of mRNA is established by the placement of the AUG start codon and initiator tRNA in the ribosomal peptidyl (P) site. Internal ribosome entry sites (IRESs) promote translation of mRNAs in a capindependent manner. We report two structures of the ribosomebound Taura syndrome virus (TSV) IRES belonging to the family of Dicistroviridae intergenic IRESs. Intersubunit rotational states differ in these structures, suggesting that ribosome dynamics play a role in IRES translocation. Pseudoknot I of the IRES occupies the ribosomal decoding center at the aminoacyl (A) site in a manner resembling that of the tRNA anticodon-mRNA codon. The structures reveal that the TSV IRES initiates translation by a previously unseen mechanism, which is conceptually distinct from initiator tRNA-dependent mechanisms. Specifically, the ORF of the IRES-driven mRNA is established by the placement of the preceding tRNA-mRNA-like structure in the A site, whereas the 40S P site remains unoccupied during this initial step.tRNA-mRNA mimicry | IRES-dependent initiation | factor-independent initiation | FREALIGN | real-space refinement

Section: Tsv Ires Stabilizes Two Partially Rotated Conformations Of Thementioning

confidence: 82%

Section: Tsv Ires Stabilizes Two Partially Rotated Conformations Of Thementioning

confidence: 99%

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Taura syndrome virus IRES initiates translation by binding its tRNA-mRNA–like structural element in the ribosomal decoding center

Koh

Brilot

Grigorieff

et al. 2014

112

“…During translocation, movement of the tRNAs into the A/P and P/ E hybrid states is accompanied by intersubunit rotational movement of the ribosome (40,41). To avoid steric clash, the A site tRNA can enter the A/P hybrid state only if the P site tRNA moves into the P/E hybrid state.…”

Section: Resultsmentioning

confidence: 99%

Crystal structures of complexes containing domains from two viral internal ribosome entry site (IRES) RNAs bound to the 70S ribosome

Zhu

Коростелев²,

Costantino³

et al. 2011

Self Cite

Internal ribosome entry site (IRES) RNAs are elements of viral or cellular mRNAs that bypass steps of canonical eukaryotic capdependent translation initiation. Understanding of the structural basis of IRES mechanisms is limited, partially due to a lack of highresolution structures of IRES RNAs bound to their cellular targets. Prompted by the universal phylogenetic conservation of the ribosomal P site, we solved the crystal structures of proposed P site binding domains from two intergenic region IRES RNAs bound to bacterial 70S ribosomes. The structures show that these IRES domains nearly perfectly mimic a tRNA•mRNA interaction. However, there are clear differences in the global shape and position of this IRES domain in the intersubunit space compared to those of tRNA, supporting a mechanism for IRES action that invokes hybrid state mimicry to drive a noncanonical mode of translocation. These structures suggest how relatively small structured RNAs can manipulate complex biological machines.ribosome structure | RNA structure | tRNA mimicry

“…Using ribosomes with deacylated tRNA in the P site and vacant A site, it was shown that ribosome binding of EF-G with a nonhydrolyzable GTP analog promoted a ratchet-like motion of the subunits and a movement of the tRNA into the P/E hybrid position (7). However, bulk FRET studies suggested that hybrid states coupled to the ratchet-like motion may form spontaneously, i.e., without binding EF-G (9,10). Further evidence for spontaneous ratcheting was provided by single-molecule FRET studies, which showed individual pretranslocation ribosomes oscillating between the nonrotated and the rotated states (4).…”

mentioning

confidence: 99%

Structure of ratcheted ribosomes with tRNAs in hybrid states

Julián

Konevega

Scheres³

et al. 2008

164

169

During protein synthesis, tRNAs and mRNA move through the ribosome between aminoacyl (A), peptidyl (P), and exit (E) sites of the ribosome in a process called translocation. Translocation is accompanied by the displacement of the tRNAs on the large ribosomal subunit toward the hybrid A/P and P/E states and by a rotational movement (ratchet) of the ribosomal subunits relative to one another. So far, the structure of the ratcheted state has been observed only when translation factors were bound to the ribosome. Using cryo-electron microscopy and classification, we show here that ribosomes can spontaneously adopt a ratcheted conformation with tRNAs in their hybrid states. The peptidyl-tRNA molecule in the A/P state, which is visualized here, is not distorted compared with the A/A state except for slight adjustments of its acceptor end, suggesting that the displacement of the A-site tRNA on the 50S subunit is passive and is induced by the 30S subunit rotation. Simultaneous subunit ratchet and formation of the tRNA hybrid states precede and may promote the subsequent rapid and coordinated tRNA translocation on the 30S subunit catalyzed by elongation factor G.translocation ͉ elongation factor G ͉ cryo-electron microscopy D uring the elongation cycle, the tRNAs⅐mRNA complex is translocated to allow reading of the following codon on mRNA. Translocation is promoted by elongation factor G (EF-G) and accompanied by GTP hydrolysis. During translocation, the ribosome changes from the pretranslocation state with deacylated tRNA in the peptidyl site (P site) and peptidyltRNA in the aminoacyl site (A site) to the posttranslocation state where A-and P-site tRNAs have moved to P and exit (E) sites, respectively. The universal design of ribosomes from two subunits inspired early suggestions that translocation may involve a movement of the subunits relative to each other (1, 2). Such models imply the existence of intermediate states for the tRNAs that differ from the classic A, P, and E states. Chemical probing experiments with pretranslocation ribosomes indicated that after peptidyl transfer the acceptor ends of the tRNAs spontaneously moved on the large 50S subunit toward their posttranslocation positions (3), indicating that peptidyl-tRNA and deacylated tRNA entered hybrid A/P and P/E states, respectively. The transition was observed in the absence of EF-G, and the driving force for the movement was attributed to different affinities of the A, P, and E sites on the 50S subunit (4) for the chemically different acceptor ends of the tRNAs (5, 6). Nevertheless, cryo-electron microscopy (cryoEM) of ribosomes in the pretranslocation state showed the tRNAs in their classic A, P, and E states (7). Although this result was difficult to reconcile with the hybrid-state model, it is important to note that in that complex the occupancy of the E site by deacylated tRNA may have prevented the P-site tRNA to enter the P/E-site hybrid state.CryoEM of ribosomes in complex with EF-G revealed a relative rotation between subunits referred to as ratc...