Altered tRNA dynamics during translocation on slippery mRNA as determinant of spontaneous ribosome frameshifting

Abstract

When reading consecutive mRNA codons, ribosomes move by exactly one triplet at a time to synthesize a correct protein. Some mRNA tracks, called slippery sequences, are prone to ribosomal frameshifting, because the same tRNA can read both 0- and –1-frame codon. Using smFRET we show that during EF-G-catalyzed translocation on slippery sequences a fraction of ribosomes spontaneously switches from rapid, accurate translation to a slow, frameshifting-prone translocation mode where the movements of peptidyl- and deacylated tRNA become uncoupled. While deacylated tRNA translocates rapidly, pept-tRNA continues to fluctuate between chimeric and posttranslocation states, which slows down the re-locking of the small ribosomal subunit head domain. After rapid release of deacylated tRNA, pept-tRNA gains unconstrained access to the –1-frame triplet, resulting in slippage followed by recruitment of the –1-frame aa-tRNA into the A site. Our data show how altered choreography of tRNA and ribosome movements reduces the translation fidelity of ribosomes translocating in a slow mode.

Decoding and Recoding of mRNA Sequences by the Ribosome

“…At the same time, the P-site tRNA moves through the E site and dissociates from the ribosome. This opens a time window for the peptidyl-tRNA to select the favorable -1-frame unhindered by the deacylated tRNA interactions with the preceding codon (96). These data demonstrate the importance of the coupled movements of tRNAs and EF-G for rapid and accurate translocation.…”

“…Notably, there is a clear correlation between the rate of translocation and frameshifting, which was used to estimate the lower limit for the rate of spontaneous frameshifting, approximately 1-10 s −1 (93). Recent smFRET experiments provide an even more detailed picture (96). On a slippery sequence, some ribosomes in the population switch from rapid, accurate translation to a slow mode in which the movements of the tRNAs in the A and P sites are uncoupled.…”

“…Force measurements suggest that ribosomes switch from the fast to the slow gear to unwind mRNA hairpins (24). However, there is a marked difference between the resulting slow gears induced by an mRNA hairpin and a slippery sequence (96). Unwinding of the hairpin slows down a very early step of translocation upon which the SSU head domain rotates forward prior to tRNA movement into the CHI state (24).…”

“…Ribosomes proceed in reversible subcodon steps against the obstacle, suggesting that they exploit fluctuations on the mRNA to overcome the mechanical barrier (24,97). In contrast, frameshifting is induced at the late steps of translocation, when the tRNA moves between the CHI and POST states and the SSU head domain moves backward; notably, slowing down the steps before CHI formation does not result in frameshifting (96). Thus, while switching gears seems to be a general ribosome response, the timing of the slow step defines the translational outcome.…”

Modulation of translational decoding by m6A modification of mRNA

“…While there is a clear effect of m 6 modification on GTP hydrolysis and dipeptide formation, the model explaining these effects by a slower association rate of the TC to the ribosome 21 is clearly not supported by our experimental data. This prompted us to examine the mechanism of decoding in more detail using smFRET approaches [32][33][34][35] comparing AAA and m 6 AAA codons, which gave the largest rate differences. smFRET experiments are designed to monitor binding and unbinding of TC to and from the ribosome, as well as movement of aa-tRNA through the ribosome, accommodation and the dynamics of the peptidyl-tRNA after peptide bond formation 32,33 .…”