Identification of endogenous SsrA-tagged proteins reveals tagging at positions corresponding to stop codons

The SsrA.SmpB quality control system adds a C-terminal degradation peptide (AANDENYALAA) to nascent chains on stalled ribosomes, thereby freeing the r ibosome and ensuring proteolysis of the tagged protein. An SsrA mutant with the tag sequence AANDEHHHHHH was used to slow degradation and facilitate, Ni2+-nitrilotriacetic acid affinity purification. Display of affinity-purif ied Escherichia coli proteins on two-dimensional gels revealed small quanti ties of a diverse set of SsrA-H-6-tagged proteins, and mass spectroscopy id entified LaeI repressor, lambda cI repressor, YbeL, GalE, RbsK, and a SlyD- kan(R) fusion protein as members of this set. For A repressor and YbeL, the SsrA-H-6 tag was added after the natural C terminus of the protein, sugges ting that tagging occurred while the ribosome idled at the termination codo n of these genes. Potential causes of tagging for the other proteins includ e interference from translation of downstream reading frames, rare codons, and gene disruption. These and previous results support a broad role for th e SsrA.SmpB system in freeing stalled ribosomes and in directing degradatio n of the products of these frustrated protein synthesis reactions.