Translation termination on mRNAs lacking a stop codon

Translation of mRNAs often terminates at the end of the coding sequence when a stop codon is encountered. However, in nonstop mRNAs that lack a stop codon, the ribosome stalls at the 3′ end of the mRNA and needs to be rescued. In bacteria, stalled ribosomes are rescued by the transfer‐messenger (tmRNA)‐SmpB, the alternative rescue factor A (ArfA)‐RF2 or ArfB proteins (1–3). Although crystal structures of ribosomes complexed with tmRNA‐SmpB and ArfB were solved, the mechanism and molecular interactions involved in the ArfA‐RF2 pathway remained unclear. Recently, an induced‐fit mechanism has been reported for this pathway (4). Here we present a cryoEM structure of the Escherichia coli 70S ribosome on a nonstop mRNA in the presence of ArfA and methylated RF2 (5). Molecular interactions revealed by the structure show how ArfA recognizes a truncated mRNA and recruits the canonical termination factor RF2 to rescue the stalled ribosome (Figure 1). The positively charged C terminus of ArfA occupies the empty mRNA entry channel on the 30S subunit. Consistent with our kinetic and biochemical data, specific molecular interactions show how ArfA recruits RF2, not RF1, to the stalled ribosome. The N‐terminal domain of ArfA interacts with the switch loop of RF2, directing the universally conserved methylated Gly‐Gly‐Glnm (GGQm) motif of RF2 into the peptidyl‐transferase center (PTC) of the ribosome. Furthermore, the conformation of the methylated GGQm motif was determined, which shows that the post‐translational modification stabilizes the side chain of glutamine and thus helps orient the carbonyl oxygen of the glutamine towards the catalytic water molecule. Taken together, we determined the molecular mechanism and interactions important for the ArfA/RF2‐mediated ribosome rescue on nonstop mRNAs and provide insights into the function of the methyl group on the GGQ motif in translation termination on the ribosome.Support or Funding InformationThis study was supported from the National Institute of General Medical Sciences of the NIH (R01‐GM120552). The computational part of the study was supported by the National Institute of General Medical Sciences (P41‐GM104601 to J.C.P. and E.T.).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.