Molecular determinants of release factor 2 for ArfA-mediated ribosome rescue

• Published in: The Journal of biological chemistry Vol. 295; no. 38; pp. 13326 - 13337
• Main Authors: Kurita, Daisuke, Abo, Tatsuhiko, Himeno, Hyouta
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 18.09.2020; American Society for Biochemistry and Molecular Biology
• Subjects: ArfA; Escherichia coli - genetics; Escherichia coli - metabolism; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Peptide Chain Termination, Translational; Peptide Termination Factors - genetics; Peptide Termination Factors - metabolism; peptidyl-tRNA hydrolysis; protein synthesis; Protein Synthesis and Degradation; RF2; ribosome; ribosome rescue; Ribosomes - genetics; Ribosomes - metabolism; RNA-Binding Proteins - genetics; RNA-Binding Proteins - metabolism; stalled ribosome; stop codon-independent termination; translation; translation control; translation release factor; ArfA; ribosome rescue; stalled ribosome; translation release factor; translation; ribosome; protein synthesis; stop codon-independent termination; translation control; peptidyl-tRNA hydrolysis; RF2
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.RA120.014664

Translation termination in bacteria requires that the stop codon be recognized by release factor RF1 or RF2, leading to hydrolysis of the ester bond between the peptide and tRNA on the ribosome. As a consequence, normal termination cannot proceed if the translated mRNA lacks a stop codon. In Escherichia coli, the ribosome rescue factor ArfA releases the nascent polypeptide from the stalled ribosome with the help of RF2 in a stop codon–independent manner. Interestingly, the reaction does not proceed if RF1 is instead provided, even though the structures of RF1 and RF2 are very similar. Here, we identified the regions of RF2 required for the ArfA-dependent ribosome rescue system. Introduction of hydrophobic residues from RF2 found at the interface between RF2 and ArfA into RF1 allowed RF1 to associate with the ArfA-ribosome complex to a certain extent but failed to promote peptidyl-tRNA hydrolysis, whereas WT RF1 did not associate with the complex. We also identified the key residues required for the process after ribosome binding. Our findings provide a basis for understanding how the ArfA-ribosome complex is specifically recognized by RF2 and how RF2 undergoes a conformational change upon binding to the ArfA-ribosome complex.