Identification of Endogenous SsrA-tagged Proteins Reveals Tagging at Positions Corresponding to Stop Codons

Abstract

The SsrA⅐SmpB quality control system adds a C-terminal degradation peptide (AANDENYALAA) to nascent chains on stalled ribosomes, thereby freeing the ribosome and ensuring proteolysis of the tagged protein. An SsrA mutant with the tag sequence AANDEHHHHHH was used to slow degradation and facilitate Ni 2؉ -nitrilotriacetic acid affinity purification. Display of affinitypurified Escherichia coli proteins on two-dimensional gels revealed small quantities of a diverse set of SsrA-H 6 -tagged proteins, and mass spectroscopy identified LacI repressor, cI repressor, YbeL, GalE, RbsK, and a SlyD-kan R fusion protein as members of this set. For repressor and YbeL, the SsrA-H 6 tag was added after the natural C terminus of the protein, suggesting that tagging occurred while the ribosome idled at the termination codon of these genes. Potential causes of tagging for the other proteins include interference from translation of downstream reading frames, rare codons, and gene disruption. These and previous results support a broad role for the SsrA⅐SmpB system in freeing stalled ribosomes and in directing degradation of the products of these frustrated protein synthesis reactions.

HemK, a class of protein methyl transferase with similarity to DNA methyl transferases, methylates polypeptide chain release factors, and hemK knockout induces defects in translational termination

“…In particular, the amount of several tagged protein species, which were also tagged in the hemK ϩ strains, is markedly increased. Because some of the major endogenous targets of tmRNA were tagged at their natural stop codons (21), this result may indicate that tagging at these sites is enhanced by a termination deficiency. The function of HemK identified here raises the question of how it leads to the suppression of the light sensitivity of a protoporphyrin IX-accumulating mutant (hemH) (1).…”

Proline Residues at the C Terminus of Nascent Chains Induce SsrA Tagging during Translation Termination

“…However, RbsK tagging depends in large part on the scarcity of the rare tRNA whereas full-length YbeL tagging is largely dependent on the chemical nature of the C-terminal residues of the nascent chain. Full-length tagging of the cI repressor (12) and UDP-galactose-4-epimerase 2 have also been observed but neither of these systems involves a C-terminal proline or a rare C-terminal codon. Perhaps other C-terminal amino acid sequences can also influence tagging via interactions with release factors or SsrA.…”

“…Because termination of translation is a relatively slow process compared with translation elongation (11), SsrA and protein release factors probably compete for binding to the A-site, while the ribosome idles at an inefficient stop codon. However, SsrA tagging of full-length proteins encoded by genes with efficient stop signals has also been observed (12). For example, the intact E. coli YbeL protein is tagged (12) even though the ybeL coding region ends with an extended UAAU stop signal which is thought to be the most efficient translation termination signal in E. coli (13,14).…”

“…However, SsrA tagging of full-length proteins encoded by genes with efficient stop signals has also been observed (12). For example, the intact E. coli YbeL protein is tagged (12) even though the ybeL coding region ends with an extended UAAU stop signal which is thought to be the most efficient translation termination signal in E. coli (13,14).…”

Proteomic identification of tmRNA substrates

“…Previous studies identified six E. coli proteins (LacI, RbsK, GalE, YbeL, PhoP, and RpsG) (28,38) and eight B. subtilis proteins (EF-Tu, FolA, GsiB, TreP, PerR, YqaA, YtoQ, and YloN) (47) that are tagged by tmRNA under normal growth conditions. Only seven of these proteins are encoded in the C. crescentus genome, and only EF-Tu was identified as a tmRNA substrate.…”