ArfA Recruits RF2 into Stalled Ribosomes |
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Authors: | Yoshihiro Shimizu |
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Institution: | Laboratory for Cell-Free Protein Synthesis, Quantitative Biology Center (QBiC), RIKEN, 2-2-3, Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650‐0047, Japan |
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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. |
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Keywords: | ArfA alternative ribosome rescue factor RF1 release factor 1 RF2 release factor 2 RF3 release factor 3 |
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