Ribosome rescue by Escherichia coli ArfA (YhdL) in the absence of trans-translation system

Abstract

SummaryAlthough SsrA(tmRNA)-mediated trans-translation is thought to maintain the translation capacity of bacterial cells by rescuing ribosomes stalled on messenger RNA lacking an in-frame stop codon, single disruption of ssrA does not crucially hamper growth of Escherichia coli. Here, we identified YhdL (renamed ArfA for alternative ribosome-rescue factor) as a factor essential for the viability of E. coli in the absence of SsrA. The ssrA-arfA synthetic lethality was alleviated by SsrA DD , an SsrA variant that adds a proteolysis-refractory tag through trans-translation, indicating that ArfA-deficient cells require continued translation, rather than subsequent proteolysis of the truncated polypeptide. In accordance with this notion, depletion of SsrA in the DarfA background led to reduced translation of a model protein without affecting transcription, and puromycin, a codonindependent mimic of aminoacyl-tRNA, rescued the bacterial growth under such conditions. That ArfA takes over the role of SsrA was suggested by the observation that its overexpression enabled detection of the polypeptide encoded by a model non-stop mRNA, which was otherwise SsrA-tagged and degraded. In vitro, purified ArfA acted on a ribosomenascent chain complex to resolve the peptidyl-tRNA. These results indicate that ArfA rescues the ribosome stalled at the 3Ј end of a non-stop mRNA without involving trans-translation.

trans-translation-mediated tight regulation of the expression of the alternative ribosome-rescue factor ArfA in Escherichia coli

“…ArfA-FL mRNA and protein were detected in much higher amounts in the ΔssrA background than in the wild type background. This is consistent with the previous observation that both nonstop mRNA and its translation product are stable in the ΔssrA background (Chadani et al, 2010; also see Keiler, 2008 for review). In the ssrA DD background, where trans-translation products will receive a proteolysisrefractory "DD-tag" and escapes from degradation, DDtagged ArfA was detected ( Fig.…”

“…This suggests that the ribosome rescue function is more critical than the protein quality control function at least in E. coli. We have recently reported that E. coli ArfA also rescues stalled ribosome in the manner distinct from transtranslation (Chadani et al, 2010). ArfA-mediated ribosome rescue is not accompanied with degradation of nascent polypeptide.…”

SmpB as the handyman of tmRNA during trans-translation

“…Recently, YhdL (or ArfA for alternative ribosome-rescue factor) was identified as an essential protein for E. coli viability in the absence of tmRNA, ArfA taking over the role of the 'tmRNA-SmpB' complex. 78 Interestingly, ArfA lacks the catalytic residues of the peptide release factors, therefore requiring an additional ligand able to hydrolyze the bond between the tRNA and the nascent peptide, possibly the Pth. In addition to, the ArfA system may be required during different physiological conditions than transtranslation or, alternatively, when the 'tmRNA-SmpB' qualitycontrol mechanism is outreached.…”

The role of SmpB and the ribosomal decoding center in licensing tmRNA entry into stalled ribosomes

“…GST levels were also monitored to control for protein expression, loading, and blotting. The GST levels are not expected to vary; in the absence of tmRNA activity, ribosomes stalled on the GST template are presumably released by an alternative rescue pathway involving ArfA, allowing GST synthesis by other ribosomes to continue (Chadani et al 2010).…”