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SUMMARY Nearly half of the ribosomes translating a particular bacteriophage T4 mRNA bypass 50 nucleotides in its middle, resuming translation 3’ of this region. How this large-scale, specific hop occurs, and what determines whether a ribosome bypasses, remains unclear. We apply single-molecule fluorescence with zero-mode waveguides to track individual Escherichia coli ribosomes during translation of T4's gene 60 mRNA. Ribosomes that bypass are characterized by a 10- to 20-fold longer pause in a non-canonical rotated state at the take-off codon. During the pause, mRNA secondary structure rearrangements are coupled to ribosome forward movement, facilitated by nascent peptide interactions that disengage the ribosome anticodon-codon interactions for slippage. Close to the landing site, the ribosome then scans the mRNA in search of the optimal base-pairing interactions. Our results provide a mechanistic and conformational framework for bypassing, highlighting a non-canonical ribosomal state to allow for mRNA structure refolding to drive large-scale ribosome movements.

Half the ribosomes translating the mRNA for phage T4 gene 60 topoisomerase subunit bypass a 50 nucleotide coding gap between codons 46 and 47. The pairing of codon 46 with its cognate peptidyl-tRNA anticodon dissociates, and following mRNA slippage, peptidyl-tRNA re-pairs to mRNA at a matched triplet 5 0 adjacent to codon 47, where translation resumes. Here, in studies with gene 60 cassettes, it is shown that the peptidyltRNA anticodon does not scan the intervening sequence for potential complementarity. However, certain coding gap mutants allow peptidyl-tRNA to scan sequences in the bypassed segment. A model is proposed in which the coding gap mRNA enters the ribosomal A-site and forms a structure that precludes peptidyl-tRNA scanning of its sequence. Dissipation of this RNA structure, together with the contribution of 16S rRNA anti-Shine-Dalgarno sequence pairing with GAG, facilitates peptidyl-tRNA repairing to mRNA. The EMBO Journal (2008) IntroductionLinear scanning from the 5 0 end of mRNAs by 40S ribosomal subunits seeking translation start sites is a general hallmark of eukaryotic protein synthesis. Scanning by the smaller eubacterial ribosomal subunit, 30S, is less well known, but in some cases, it may move from a standby site into the final initiation site (Unoson and Wagner, 2007). Also, in some cases following termination, the 30S subunit scans with the potential to find reinitiation codons (Klovins et al, 1997;Wills et al, 1997;Jin et al, 2006). Important exceptions to the universality of linear scanning for eukaryotic translation initiation occur where there is internal ribosome entry, and also where the scanning is nonlinear. The latter, known as ribosome shunting, has been studied in decoding of some cellular mRNAs from mammals to an alga and especially in several viral mRNAs, including cauliflower mosaic virus and adenoviruses (Hemmings-Mieszczak et al, 2000;Xi et al, 2005;Babinger et al, 2006) Adenovirus shunting may involve rRNA:mRNA interactions in translating ribosomes (Yueh and Schneider, 2000;Chappell et al, 2006). Translating 'whole' ribosomes also exhibit the ability to bypass mRNA segments and resume translation at a downstream site to synthesize one polypeptide from two open reading frames (ORFs), or a polypeptide lacking internal sequence from a single ORF. Such translational bypassing was initially discovered as low-level 'error' bypassing of stop codons, 'stop hopping', in Escherichia coli (Weiss et al, 1987;O'Connor et al, 1989). In one of these cases, the 9 nt sequence CUU_UAG_CUA encoded a single amino acid, leucine. The anticodon of peptidyl-tRNA Leu dissociates from pairing with the CUU, the 'take-off' codon, and scans the mRNA as it linearly slips through the ribosome. The anticodon re-pairs to mRNA where it finds complementarity, that is, at the 3 0 CUA, the 'landing-site' codon. Standard decoding then resumes at the next codon. Short distance stop codon bypassing also occurs at the end of the b-globin mRNA of rabbits (Chittum et al, 1998). Similar bypassing involving pepti...