November 18, 2019

Translational of the take-off stem loop is highlighted in

Translational
bypassing in bacteriophage T4 gene 60

The best-studied case of
programmed bypassing is upon translation of bacteriophage
T4 gene 60 that codes for a subunit of a viral DNA topoisomerase (Huang et
al., 1988; Weiss, Huang and Dunn, 1990). Gene 60 is interrupted by 50
non-coding nucleotides that separate the first 46 codons of gene 60 from the
last 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 46
codons up to the take-off Glycine codon (GGA) followed by a stop codon(UAG),
but instead of terminating translation, the ribosome pauses and bypasses the
next 50 nucleotides of the mRNA and resumes translation after the landing
Glycine codon (Agirrezabala et al., 2017). All ribosomes initiate bypass at the
take-off GGA codon but only 50% of the ribosomes continue translation while the
rest fail to resume coding (Maldonado and Herr, 1998, Wills et al., 2008). This efficiency in bypassing (50%) was less than
previously reported (70%) (Weiss, Huang and Dunn, 1990). As a result, truncated
and full-length proteins in different ratios are produced. Much of the current
research in this field focuses on understanding the mechanism by which gene 60 mRNA
guides the ribosome toward bypassing and avoiding premature termination.  

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This project will aim at
probing the requirements for translational bypassing in gene 60 for the
development of artificial translational ‘hop’ sequences that can be safely
introduced in other genes, allowing production of truncated and full-length
proteins in different stoichiometric ratios.

 

Requirements
for translational bypass

A
particular 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 stem
loop structure 5′ of the coding gap and a stop codon UAG immediately after the
take-off codon are required for bypass (8-13) (Figure 1).

Nascent peptide signal

 

 

 

50 nt coding gap

 

 

 

Landing  

 

Figure
1.
Gene 60 mRNA and its regulatory elements required for bypass. The take-off and
landing GGA codon are shown in green. The stop codon UAG is shown in red.
The
apical part of the take-off stem loop is highlighted in gray. The signals
of the nascent peptide are shown by arrows. Adapted from (Agirrezabala e al.,
2017)

 It was believed that termination at the UAG
stop codon competes with bypassing but it was proved otherwise (Herr, Gesteland
and Atkins,2000). The presence of a stem loop 3´ of the coding gap is
debatable, since studies showed that its deletion or fusion of a reporter protein
such 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 that
the 5″ and 3″ end of mRNA gene 60 fold independently of each other, with
greater 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 elements
guide the ribosome toward bypassing and avoid premature termination and
readthrough isn’t well understood. the structure of a translating
ribosome stalled at the bypassing take-off

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