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.

-RssB-ClpXP complex forms. The complex degrades S and releases RssB from ClpXP in an ATP-dependent reaction. Our results illuminate an important mechanism for regulated protein turnover in which a unique targeting protein, whose own activity is regulated through specific signaling pathways, catalyzes the delivery of a specific substrate to a specific protease.

The product of the Escherichia coli rpoH (htpR) gene, (F32, is required for heat-inducible transcription of the heat shock genes. Previous studies on the role of &2 in growth at low temperature and in gene expression involved the use of nonsense and missense rpoH mutations and have led to ambiguous or conflicting results. To clarify the role of c32 in cell physiology, we have constructed loss-of-function insertion and deletion mutations in rpoH. Strains lacking r32 are extremely temperature sensitive and grow only at temperatures less than or equal to 20°C. There is no transcription from the heat shock promoters preceding the htpG gene or the groESL and dnaKJ operons; however, several heat shock proteins are produced in the mutants. GroEL protein is present in the rpoH null mutants, but its synthesis is not inducible by a shift to high temperature. The low-level synthesis of GroEL results from transcription initiation at a minor tT70-controlled promoter for the groE operon. DnaK protein synthesis cannot be detected at low temperature, but can be detected after a shift to 42°C. The mechanism of this heat-inducible synthesis is not known. We conclude that cr32 iS required for cell growth at temperatures above 20°C and is required for transcription from the heat shock promoters. Several heat shock proteins are synthesized in the absence of r32, indicating that there are additional mechanisms controlling the synthesis of some heat shock proteins.When cells or organisms are suddenly exposed to high temperature a set of heat shock proteins are transiently induced. The response is apparently universal, having been observed in members of all phylogenetic kingdoms (reviewed in reference 19). In addition, some heat shock proteins have been conserved during evolution. The eucaryotic heat shock proteins Hsp7O and Hsp83 have nearly 50% of their amino acid residues in common with their procaryotic homologs (3, 4). The functions of the heat shock proteins are not well understood. These proteins seem to be involved in a wide variety of cellular processes and are required for prolonged survival at high temperatures (19).In Escherichia coli the heat shock response is rapidly induced. Within 1 min after a shift to high temperature, transcription initiation from the heat shock promoters increases, leading to the elevated synthesis of the heat shock proteins (reviewed in references 24 and 26). The heat shock promoters are recognized in vitro by RNA polymerase containing the 32-kilodalton a subunit (E&2) but not by RNA polymerase containing the primary 70-kilodalton u subunit (E070) (8,10,12). Genetic studies from a number of laboratories support the idea that &32, the product of the rpoH gene, is required for the increased transcription of the heat shock genes after a shift to high temperature. Strains carrying supC(Ts), encoding a temperature-sensitive suppressor tRNA, and the rpoH165 amber mutation fail to induce the synthesis of heat shock proteins and are temperature sensitive for growth (23,26,46 tration of &2 in the cell and t...

The DnaK, DnaJ, and GrpE heat shock proteins are required for motility of Escherichia coli. Cells deleted for dnaK or dnaJ, or with some mutations in the dnaK or grpE gene, are nonmotile, lack flagella, exhibit a 10- to 20-fold decrease in the rate of synthesis of flagellin, and show reduced rates of transcription of both the flhD master operon (encoding FlhD and FlhC) and the fliA operon (encoding sigma F). Genetic studies suggest that DnaK and DnaJ define a regulatory pathway affecting flhD and fliA synthesis that is independent of cyclic AMP-catabolite gene activator protein or the chemotaxis system.

RpoS, the stationary-phase sigma factor of Escherichia coli, is responsible for increased transcription of an array of genes when cells enter stationary phase and under certain stress conditions. RpoS is rapidly degraded during exponential phase and much more slowly during stationary phase; the resulting changes in RpoS accumulation play an important role in providing differential expression of RpoS-dependent gene expression. It has previously been shown that rapid degradation of RpoS during exponential growth depends on RssB (also called SprE and MviA), a protein with homology to the family of response regulators, and on the ClpXP protease. We find that RssB regulation of proteolysis does not extend to another ClpXP substrate, bacteriophage lambda O protein, suggesting that RssB acts on the specific substrate RpoS rather than on the protease. In addition, the activity of RpoS is down-regulated by RssB when degradation is blocked. In cells blocked for RpoS degradation by a mutation inclpP, cells devoid of RssB show a four- to fivefold-higher activity of an RpoS-dependent reporter fusion than cells overproducing RssB. Therefore, RssB allows specific environmental regulation of RpoS accumulation and may also modulate activity. The regulation of degradation provides an irreversible switch, while the regulation of activity may provide a second, presumably reversible level of control.