Amino acid starvation and colicin D treatment induce A-site mRNA cleavage in Escherichia coli.
Escherichia 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.
Garza-Sánchez
Fernando
F
Department of Molecular, Cellular and Developmental Biology, University of California Santa Barbara, Santa Barbara, CA 93106-9610, USA.
Gin
Jennifer G
JG
Hayes
Christopher S
CS
D000596:Amino Acids / Q000378:metabolism*; D001665:Binding Sites; D018388:Codon, Terminator; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism*; D012343:RNA, Transfer / Q000378:metabolism
D000596:Amino Acids; D018388:Codon, Terminator; D029968:Escherichia coli Proteins; D012333:RNA, Messenger; C501879:colicin D immunity protein, E coli; D012343:RNA, Transfer