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

Abstract

Ribosomes translating mRNA without an in-frame stop codon (non-stop mRNA) stall at its 3' end. In eubacteria, such ribosomes are rescued by SsrA-mediated trans-translation. Recently, we have shown that Escherichia coli ArfA (formerly YhdL) also rescues stalled ribosomes by a mechanism distinct from that of transtranslation. Synthetic lethality phenotype of ssrA arfA double mutants suggests that accumulation of stalled ribosomes is deleterious to E. coli cells. In this report, we show that the expression of ArfA is tightly regulated by the system involving trans-translation. Both premature transcription termination and specific cleavage by RNase III were programmed at the specific sites within the arfA open reading frame (ORF) and produced arfA non-stop mRNA. C-terminally truncated ArfA protein synthesized from arfA non-stop mRNA was tagged through SsrA-mediated trans-translation and degraded in wild type cell. In the absence of SsrA, however, C-terminally truncated ArfA escaped from degradation and had a function to rescue stalled ribosomes. Full-length ArfA produced only when arfA mRNA escapes from both premature transcription termination and RNase III cleavage was unstable. From these results, we illustrate a regulatory model in which ArfA is expressed only when it is needed, namely, when the ribosome rescue activity of trans-translation system is insufficient to support cell viability. This sophisticated regulatory mechanism suggests that the ArfA-mediated ribosome rescue is a backup system for trans-translation.

Mechanism of ribosome rescue by ArfA and RF2

“…This suggests conformational disorder of the C-terminus at the entrance to the mRNA tunnel. Our structures are consistent with biochemical studies (Chadani et al, 2011), which showed that ArfA is functional with a C-terminal truncation at Asn47, but further shortening inactivates ArfA. In particular, truncations following Met40—removing at least five basic amino acids that bind in the tunnel—abrogate ArfA-mediated release by reducing ArfA affinity for the 70S ribosome (Chadani et al, 2011).…”

“…[Keiler, 2015]). The ArfA ( a lternative r escue f actor A ) pathway is essential in trans-translation-deficient cells (Chadani et al, 2010) and is thought to function as a backup mechanism for trans-translation in enterobacteria (Garza-Sánchez et al, 2011; Chadani et al, 2011; Schaub et al, 2012). ArfA is a small protein (~70 aa in most organisms), with only 47 amino acids sufficient for function, as shown for E. coli ArfA truncations (Garza-Sánchez et al, 2011).…”

A ribosome profiling study of mRNA cleavage by the endonuclease RelE

“…The levels of synthesis of the ArfA protein (assessed by ribosome occupancy) are more than 80-fold increased. Interestingly, ArfA expression is regulated by tmRNA in an elegant feedback mechanism such that when the capacity of tmRNA is exceeded, ArfA accumulates (29,30). The fact that ArfA is strongly upregulated in cells treated with RelE suggests that the tmRNA system is overwhelmed and that cells are responding to the need for more ribosome rescue activity by upregulating ArfA expression.…”

ArfA Recruits RF2 into Stalled Ribosomes

“…Interestingly, the expression of ArfA is tightly regulated by the system involving transtranslation. 21 This suggests that the ArfAdependent system may play a backup role to the tmRNA-dependent system. Additionally, the tmRNA rescue system is linked to the SspB-ClpXP protein degradation system for the maintenance of the quality of cellular proteins, 10 which suggests a more prevailing role for the tmRNA rescue system in the translation maintenance.…”