Translational recoding: canonical translation mechanisms reinterpreted

Abstract

During canonical translation, the ribosome moves along an mRNA from the start to the stop codon in exact steps of one codon at a time. The collinearity of the mRNA and the protein sequence is essential for the quality of the cellular proteome. Spontaneous errors in decoding or translocation are rare and result in a deficient protein. However, dedicated recoding signals in the mRNA can reprogram the ribosome to read the message in alternative ways. This review summarizes the recent advances in understanding the mechanisms of three types of recoding events: stop-codon readthrough, -1 ribosome frameshifting and translational bypassing. Recoding events provide insights into alternative modes of ribosome dynamics that are potentially applicable to other non-canonical modes of prokaryotic and eukaryotic translation.

Figure 1.

Three types of recoding events. Translational readthrough extends the polypeptide C-terminally allowing the production of two protein isoforms from the same transcript. Frameshifting produces typically two functional polypeptides from different reading frames of the same mRNA. Bypassing is a recoding event that synthesizes one protein from two open discontinuous reading frames.

Figure 2.

Factors affecting translational readthrough in eukaryotes. Cis factors that affect readthrough include sequences upstream of the stop codon (light gray), the identity of the stop codon (red-orange), the +4 nucleotide (blue) and the downstream sequences that occupy the mRNA channel (green). Distal cis element includes downstream mRNA secondary structure. Among several trans factors that affect readthrough, the specific case of hnRNP A2/B1 is depicted. hnRNP A2/B1 promotes readthrough by binding to a cis element in the 3′ UTR of mammalian gene VEGFA. A, P and E depict the three stable tRNA-binding sites. SSU, small ribosomal subunit; LSU, large ribosomal subunit.

Figure 3.

UGA recoding by Sec-tRNA^Sec. (A) Structure of the SelB–GTP–Sec-tRNA^Sec complex on the ribosome during recoding (modified from (68)). The GTPase of SelB is activated by the sarcin–ricin loop (SRL) of 23S rRNA. (B) SECIS-mediated Sec insertion versus RF2-dependent termination at UGA. The Sec-tRNA^Sec–SelB–GTP complex is rapidly recruited to the SECIS while still distant from the ribosome. Step 1: while the ribosome moves along the mRNA toward the UGA codon, the lower part of the SECIS becomes unwound and the Sec-tRNA^Sec–SelB–GTP complex occupies the entry to the A site, thereby hindering the recruitment of RF2 to the stop codon. Step 2: after delivery of Sec-tRNA^Sec to the A site and Sec insertion into the growing peptide, the ribosome can recruit the next EF-Tu–GTP–aa-tRNA complex (gray) and continue translation. Alternatively (step 3), if Sec incorporation fails, the A site becomes accessible for RF2, which promotes termination and peptide release.

Figure 4.

Reading frame maintenance during translocation. Positions of the P- and A-site tRNAs in the intermediate state of translocation in the presence and absence of EF-G (left panel) and the schematics illustrating the movement of the tRNA anticodons toward the –1-frame in the absence of EF-G (right panel) (reproduced from Zhou, J., Lancaster, L., Donohue, J.P. and Noller, H.F. (2019) Spontaneous ribosomal translocation of mRNA and tRNAs into a chimeric hybrid state. Proc. Natl. Acad. Sci. U.S.A., 116, 7813–7818 (78) with permission). Complexes depicted in the schematics are from (79) with EF-G and from (78) without EF-G and contain a different sets of tRNAs in the A and P sites.

Figure 5.

Mechanism of –1PRF. Under conditions where aa-tRNAs are abundant (blue box), –1PRF takes place during the late stage of translocation by two-tRNA slippage (the P- and A-site tRNAs are shown in magenta and blue, respectively). Aa-tRNA limitation (yellow box) causes translational pausing that leads to the one-tRNA slippage of the P-site tRNA. X XXY YYZ is the sequence of the slippery site; abc and Zab are codons following the slippery site in 0- and –1-frames, respectively.

Figure 6.

Translational bypassing. (A) Schematic of gene 60 mRNA. The nascent peptide, the SL element upstream (5′ SL) and downstream (3′ SL) of the take-off site, as well as the take-off SL are key elements facilitating bypassing. (B) Structure of the nascent peptide in the exit tunnel of the ribosome (left panel) and of the short A-site SL (right panel) obtained by cryo-EM (133). Middle panel represents a cartoon of the ribosome at the take-off site. (C) Schematic of bypassing. For details, see text. Rotation of the SSU relative to the LSU is indicated by different shades of blue. The blurred cartoon represents the ribosome in motion.