Amino Acid Starvation and Colicin D Treatment Induce A-site mRNA Cleavage in Escherichia coli

Abstract

SUMMARYEscherichia coli possesses a unique RNase activity that cleaves stop codons in the ribosomal aminoacyl-tRNA binding site (A-site) during inefficient translation termination. This A-site mRNA cleavage allows recycling of arrested ribosomes by facilitating recruitment of the tmRNA•SmpB ribosome rescue system. To test whether A-site nuclease activity also cleaves sense codons, we induced ribosome pausing at each of the six arginine codons using three strategies -rare codon usage, arginine starvation, and inactivation of arginine tRNAs with colicin D. In each instance, ribosome pausing induced mRNA cleavage within the target arginine codons, and resulted in tmRNA-mediated SsrA-peptide tagging of the nascent polypeptide. A-site mRNA cleavage did not require the stringent factor (ppGpp), or bacterial toxins such as RelE, which mediates a similar nuclease activity. However, the efficiency of A-site cleavage was modulated by the identity of the two codons immediately upstream (5′ side) of the A-site codon. Starvation for histidine and tryptophan also induced A-site cleavage at histidine and tryptophan codons, respectively. Thus, A-site mRNA cleavage is a general response to ribosome pausing, capable of cleaving a variety of sense and stop codons. The induction of A-site cleavage during amino acid starvation suggests this nuclease activity may help to regulate protein synthesis during nutritional stress.

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

“…Shortage of amino acids or tRNA causes ribosome stall during translation (Garza-Sánchez et al, 2008;Li et al, 2008). Ribosome stalling induces mRNA breakage and produces the target of ribosome rescue (Sunohara et al, 2004).…”

Resolving Nonstop Translation Complexes Is a Matter of Life or Death

“…For example, in some substrates, tagging occurs with high frequency after runs of rare codons or highly inefficient translation termination sequences (40)(41)(42). The mRNA is initially complete in these cases, but ribosome stalling during translation elongation or termination exposes the downstream mRNA to exonucleases, which chew back the mRNA to the leading edge of the ribosome to generate substrates for trans-translation (4,(43)(44)(45). Exonuclease activity by RNase II can promote cleavage of the mRNA in the A site through an unknown mechanism, but RNase II and the corresponding A-site cleavage are not essential for trans-translation on known substrates (46,47).…”

Rejection of tmRNA·SmpB after GTP hydrolysis by EF-Tu on ribosomes stalled on intact mRNA

“…In contrast, several in vivo studies seemed to suggest that trans-translation occurs even in the middle of mRNA at several contiguous rare codons, inefficient stop codons, and certain nascent peptide sequences, all of which pause or arrest translation Sauer 1999, 2001;Collier et al 2002;Hayes et al 2002). One solution to this paradox is that, regardless of the mechanism, prolonged translational arrest in the middle of an mRNA induces endonucleolytic cleavage in or around the A-site codon, generating nonstop mRNA (Hayes and Sauer 2003;Sunohara et al 2004;Li et al 2007;Garza-Sánchez et al 2008). On the other hand, some in vivo studies observe robust tmRNA activity on transcripts with 15-21 nt downstream from the P-site codon, following degradation of the mRNA by 3 ′ -5 ′ exonucleases to the 3 ′ boundary protected by the ribosome (Garza-Sánchez et al 2009;Janssen et al 2013).…”