title: ArfA recruits RF2 into stalled ribosomes.
abstract: During translation in Escherichia coli, the ribosome rescue factor YaeJ and the alternative ribosome rescue factor (ArfA, previously called YhdL) can release stalled ribosomes from mRNA. Here, I used a reconstituted cell-free protein synthesis system to examine YaeJ- and ArfA-dependent recycling of stalled ribosomes, in which mRNA lacks in-frame stop codons. It is shown that YaeJ alone could recycle the ribosome but that ArfA required the presence of release factor 2 (RF2). Furthermore, I show that RF2 binds to stalled ribosomes only in the presence of ArfA, demonstrating that ArfA recruits RF2 into the A site of the ribosome to facilitate peptidyl-tRNA hydrolysis. It is also demonstrated that the efficiency of the ArfA-dependent process decreases rapidly with an increase in mRNA length downstream of the A site of the ribosome whereas YaeJ function is maintained on mRNA with sufficient length. From the results, I discuss differences of in vivo roles of these two systems in addition to the well-known tmRNA-dependent trans-translation system.
authors.affiliation: Laboratory for Cell-Free Protein Synthesis, Quantitative Biology Center (QBiC), RIKEN, 2-2-3, Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan. yshimizu@riken.jp
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Shimizu
mesh_terms: D002265:Carboxylic Ester Hydrolases / Q000378:metabolism*; D002474:Cell-Free System; D018388:Codon, Terminator / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D010454:Peptide Termination Factors / Q000378:metabolism*; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics; D016601:RNA-Binding Proteins / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*
chemical_list: C556014:ArfA protein, E coli; D018388:Codon, Terminator; D029968:Escherichia coli Proteins; D010454:Peptide Termination Factors; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; C489006:prfB protein, E coli; D002265:Carboxylic Ester Hydrolases; C558457:YaeJ protein, E coli