SsrA-mediated peptide tagging caused by rare codons and tRNA scarcity

SsrA RNA mediates the addition of a C-terminal peptide tag (AANDENYALAA) to bacterial proteins translated from mRNAs without in-frame stop codons. Thi s process involves both tRNA- and mRNA-like functions of SsrA and targets t he tagged proteins for degradation. By designing an SsrA variant that adds a peptide tag (AANDENYALDD) that does not result in rapid degradation, we s how that tagging of a model protein synthesized from an mRNA without stop c odons can be detected both in vivo and in vitro. We also use this assay to demonstrate that ribosome stalling at clusters of rare arginine codons in m RNA is sufficient to recruit and activate the SsrA peptide tagging system. An essential requirement for tagging at rare AGA codons is a scarcity of th e cognate tRNA; supplemental tRNA(AGA) suppresses tagging, and depleting th e available pool of tRNAAGA enhances tagging and reveals tagging caused by single rare AGA codons. Protein tagging at sites corresponding to rare codo ns appears to involve SsrA action at an internal mRNA site rather than at t he 3' end of a cleaved mRNA.