Variants of lambda repressor and cytochrome b562 translated from messenger RNAs without stop codons were modified by carboxyl terminal addition of an ssrA-encoded peptide tag and subsequently degraded by carboxyl terminal-specific proteases present in both the cytoplasm and periplasm of Escherichia coli. The tag appears to be added to the carboxyl terminus of the nascent polypeptide chain by cotranslational switching of the ribosome from the damaged messenger RNA to ssrA RNA.

Ribosomes that stall during translation need to be rescued to ensure that the protein synthesis capacity of the cell is maintained. Stalling arises when ribosomes become trapped at the 3' end of an mRNA, which occurs when a codon is unavailable, as this leads to the arrest of elongation or termination. In addition, various factors can induce ribosome stalling in the middle of an mRNA, including the presence of specific amino acid sequence motifs in the nascent polypeptide. Almost all bacteria use a mechanism known as trans-translation to rescue stalled ribosomes, and some species also have other rescue mechanisms that are mediated either by the alternative ribosome-rescue factor A (ArfA) or ArfB. In this Review, I summarize the recent studies that have demonstrated the conditions that trigger ribosome stalling, the pathways that bacteria use to rescue stalled ribosomes and the physiological effects of these processes.

An Escherichia coli protease designated Tsp (tail-specific protease) has been purified, and its gene has been cloned and sequenced. Tsp specifically degrades a variant of the N-terminal domain of A repressor in which the five C-terminal residues, which are polar in wild type, have been replaced by nonpolar residues. This substrate specificity in vitro parallels the previously reported selective degradation in vivo of N-terminal-domain variants with nonpolar C-terminal residues. The gene sequence and N-terminal protein sequence of Tsp predict a protein of 660 amino acids. The deduced protein sequence of Tsp shows no significant homology to known protease sequences but does show sequence similarity to the human and bovine interphotoreceptor retinoid-binding proteins, which bind hydrophobic ligands.Within cells, different proteins are degraded with half-lives ranging from minutes to days (1). This heterogeneity implies that intracellular degradation is selective, but the mechanisms of this selectivity are incompletely understood. The C-terminal sequences of some proteins can have dramatic effects on their rates of degradation in Escherichia coli (2, 3). For example, the wild-type N-terminal domain of A repressor (residues 1-102) has the polar C-terminal amino acid sequence RSEYE102 and has a half-life in vivo of >10 hr. The rapidly degraded "#105" variant of this protein has the hydrophobic C-terminal sequence WVAAA102 and has a half-life of 15 min (3). To identify possible cellular component(s) responsible for the degradation of proteins with destabilizing C termini, we have purified an E. coli activity that specifically degrades the #105 variant but not the wild-type N-terminal domain. This activity resides in a single, purified polypeptide (Tsp, for tail-specific protease). We have cloned and sequenced the tsp gene.* Based on sequence comparisons, Tsp shows similarities to the human and bovine interphotoreceptor retinoid-binding proteins (IRBPs) but does not resemble any proteases in the protein sequence data base. MATERIALS AND METHODSAssays for Tsp Activity. Tsp activity in crude lysates and column fractions was assayed by measuring the degradation of 35S-labeled substrates (either the #105 variant or the wild-type N-terminal domain of A repressor) to products soluble in 10% (wt/vol) trichloroacetic acid (3). Reaction mixtures contained _9000 cpm of 35S-labeled substrate, a sample of the E. coli fraction in lysis buffer or column buffer, and 0.02% Nonidet P-40 in a volume of 50 p1. Reaction mixtures were incubated at 370C for 1 or 2 hr and were processed as described (3).In more purified fractions, degradation was assayed by the appearance of substrate digestion products following SDS/ PAGE. Wild-type or #105 protein (2 ,ug), which was purified by the method of Lim and Sauer (4), was mixed with a sample in a 10-1LI reaction volume. Reaction mixtures were incubated at 370C for 3-6 hr and were stopped by boiling for 3 min in Laemmli sample buffer (5). Samples were electrophoresed in SDS/16.5% polyacrylam...

A general mechanism in bacteria to rescue stalled ribosomes and to clear the cell of incomplete polypeptides involves an RNA species, tmRNA (SsrA), which functions as both a tRNA and an mRNA. This RNA encodes a peptide tag that is incorporated at the end of the aberrant polypeptide and targets it for proteolysis. We have identified a circularly permuted version of the tmRNA gene in alpha-proteobacteria as well as in a lineage of cyanobacteria. The genes in these two groups seem to have arisen from two independent permutation events. As a result of the altered genetic structure, these tmRNAs are composed of two distinct RNA molecules. The mature two-piece tmRNAs are predicted to have a tRNA-like domain and an mRNA-like domain similar to those of standard one-piece tmRNAs, with a break located in the loop containing the tag reading frame. A related sequence was found in the mitochondrial genome of Reclinomonas americana, but only the tRNA-like portion is retained. Although several sequence and structural motifs that are conserved among one-piece tmRNAs have been lost, the alpha-proteobacterium Caulobacter crescentus produces a functional two-piece tmRNA.