Translational of the take-off stem loop is highlighted in

Translationalbypassing in bacteriophage T4 gene 60The best-studied case ofprogrammed bypassing is upon translation of bacteriophageT4 gene 60 that codes for a subunit of a viral DNA topoisomerase (Huang etal., 1988; Weiss, Huang and Dunn, 1990). Gene 60 is interrupted by 50non-coding nucleotides that separate the first 46 codons of gene 60 from thelast 114 codons and it was shown to trigger bypassing (Huang et al., 1988,Maldonado and Herr,1998, Todd and Walter, 2013). Upon translation of gene 60, the ribosome translates the first 46codons up to the take-off Glycine codon (GGA) followed by a stop codon(UAG),but instead of terminating translation, the ribosome pauses and bypasses thenext 50 nucleotides of the mRNA and resumes translation after the landingGlycine codon (Agirrezabala et al.

, 2017). All ribosomes initiate bypass at thetake-off GGA codon but only 50% of the ribosomes continue translation while therest fail to resume coding (Maldonado and Herr, 1998, Wills et al., 2008). This efficiency in bypassing (50%) was less thanpreviously reported (70%) (Weiss, Huang and Dunn, 1990). As a result, truncatedand full-length proteins in different ratios are produced. Much of the currentresearch in this field focuses on understanding the mechanism by which gene 60 mRNAguides the ribosome toward bypassing and avoiding premature termination.

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 This project will aim atprobing the requirements for translational bypassing in gene 60 for thedevelopment of artificial translational ‘hop’ sequences that can be safelyintroduced in other genes, allowing production of truncated and full-lengthproteins in different stoichiometric ratios. Requirementsfor translational bypassAparticular sequence in the nascent peptide, a stem loop within the coding gap,a ‘landing site’ codon identical to the ‘take-off’ codon (in this case GGA), a stemloop structure 5′ of the coding gap and a stop codon UAG immediately after thetake-off codon are required for bypass (8-13) (Figure 1). Nascent peptide signal       50 nt coding gap   3´   5´   Landing     Figure1.Gene 60 mRNA and its regulatory elements required for bypass. The take-off andlanding GGA codon are shown in green. The stop codon UAG is shown in red.

Theapical part of the take-off stem loop is highlighted in gray. The signalsof the nascent peptide are shown by arrows. Adapted from (Agirrezabala e al.,2017)  It was believed that termination at the UAGstop codon competes with bypassing but it was proved otherwise (Herr, Gestelandand Atkins,2000). The presence of a stem loop 3´ of the coding gap isdebatable, since studies showed that its deletion or fusion of a reporter proteinsuch as lacZ 2 codons after the landing codon had no effect on bypassing(Samatova et al., 2014, Weiss, Huang ang Dunn, 1990). It was also shown thatthe 5″ and 3″ end of mRNA gene 60 fold independently of each other, withgreater importance of the 5″ structures than the 3″ structures in bypassing(Todd and Walter, 2013).

Furthermore, mutations done to the nascent peptide,the take/off and landing codons, the stem loop within the coding gap and the 5″Stem loop all affected bypassing (Samatova et al., 2014, Weiss, Huang ang Dunn,1990, Herr et al., 2004). However, how these stimulatory elementsguide the ribosome toward bypassing and avoid premature termination andreadthrough isn’t well understood. the structure of a translatingribosome stalled at the bypassing take-off