Interruption of translation in Escherichia coli can lead to the addition of an 11-residue carboxy-terminal peptide tail to the nascent chain. This modification is mediated by SsrA RNA (also called 10Sa RNA and tmRNA) and marks the tagged polypeptide for proteolysis. Degradation in vivo of repressor amino-terminal domain variants bearing this carboxy-terminal SsrA peptide tag is shown here to depend on the cytoplasmic proteases ClpXP and ClpAP. Degradation in vitro of SsrA-tagged substrates was reproduced with purified components and required a substrate with a wild-type SsrA tail, the presence of both ClpP and either ClpA or ClpX, and ATP. Clp-dependent proteolysis accounts for most degradation of SsrA-tagged amino-domain substrates at 32°C, but additional proteases contribute to the degradation of some of these SsrA-tagged substrates at 39°C. The existence of multiple cytoplasmic proteases that function in SsrA quality-control surveillance suggests that the SsrA tag is designed to serve as a relatively promiscuous signal for proteolysis. Having diverse degradation systems able to recognize this tag may increase degradation capacity, permit degradation of a wide variety of different tagged proteins, or allow SsrA-tagged proteins to be degraded under different growth conditions.

SsrA RNA mediates the addition of a C-terminal peptide tag (AANDENYALAA) to bacterial proteins translated from mRNAs without in-frame stop codons. This process involves both tRNA-and mRNA-like functions of SsrA and targets the tagged proteins for degradation. By designing an SsrA variant that adds a peptide tag (AANDENYALDD) that does not result in rapid degradation, we show that tagging of a model protein synthesized from an mRNA without stop codons 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 mRNA is sufficient to recruit and activate the SsrA peptide tagging system. An essential requirement for tagging at rare AGA codons is a scarcity of the cognate tRNA; supplemental tRNA AGA suppresses tagging, and depleting the available pool of tRNA AGA enhances tagging and reveals tagging caused by single rare AGA codons. Protein tagging at sites corresponding to rare codons appears to involve SsrA action at an internal mRNA site rather than at the 3Ј end of a cleaved mRNA.

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.

FTD is a common symptom of psychosis and may be considered a marker of illness severity. Detailed dimensional assessment of FTD can clarify diagnosis and may help predict prognosis.