Cells subjected to a heat shock, or a variety of other stresses increase the synthesis of a set of proteins, known as heat shock proteins. This response is apparently universal, occurring in the entire range from bacterial to mammalian cells. In Escherichia coli heat shock protein synthesis transiently increases following a shift from 30 degrees C to 42 degrees C as a result of changes in transcription initiation at heat shock promoters. Heat shock promoters are recognized by RNA polymerase containing a sigma factor of relative molecular mass (Mr) 32,000 (32K) E sigma 32 and not E sigma 70, the major form of RNA polymerase holoenzyme. To determine whether changes in the concentration of sigma 32 regulate this response, we measured the amount of sigma 32 before and after shift to high temperature and found that it increased transiently during heat shock as a result of changes in sigma 32 synthesis and stability. Our results indicate that sigma 32 is directly responsible for regulation of the heat shock response.

The Escherichia coli DnaK heat shock protein has been identified previously as a negative regulator of E. coli heat shock gene expression. We report that two other heat shock proteins, DnaJ and GrpE, are also involved in the negative regulation of heat shock gene expression. Strains carrying defective dnaK, dnaj, or grpE alleles have enhanced synthesis of heat shock proteins at low temperature and fail to shut off the heat shock response after shift to high temperature. These regulatory defects are due to the loss of normal control over the synthesis and stability of CT^^, the alternate RNA polymerase tr-factor required for heat shock gene expression. We conclude that DnaK, DnaJ, and GrpE regulate the concentration of o-^^. We suggest that the synthesis of heat shock proteins is controlled by a homeostatic mechanism linking the function of heat shock proteins to the concentration of a^^.

No abstract

The sigma 70 subunit of E. coli RNA polymerase is required for sequence-specific recognition of promoter DNA. Genetic studies and sequence analysis have indicated that sigma 70 contains two specific DNA-binding domains that recognize the two conserved portions of the prokaryotic promoter. However, intact sigma 70 does not bind to DNA. Using C-terminal and internal polypeptides of sigma 70, carrying one or both putative DNA-binding domains, we demonstrate that sigma 70 does contain two DNA-binding domains, but that N-terminal sequences inhibit the ability of intact sigma 70 to bind to DNA. Thus, we propose that sigma 70 is a sequence-specific DNA-binding protein that normally functions through an allosteric interaction with the core subunits of RNA polymerase.

In order to analyze the in vivo role of the SSA class of cytosolic 70-kDa heat shock proteins (hsps) of Saccharomyces cerevisiae, we isolated a temperature-sensitive mutant of SSA1. The effect of a shift of mutant cells (ssa1 ts ssa2 ssa3 ssa4) from the permissive temperature of 23؇C to the nonpermissive temperature of 37؇C on the processing of several precursor proteins translocated into the endoplasmic reticulum or mitochondria was assessed. Of three mitochondrial proteins tested, the processing of only one, the ␤ subunit of the F 1 F 0 ATPase, was dramatically affected. Of six proteins destined for the endoplasmic reticulum, the translocation of only prepro-␣-factor and proteinase A was inhibited. The processing of prepro-␣-factor was inhibited within 2 min of the shift to 37؇C, suggesting a direct effect of the hsp70 defect on translocation. More than 50% of radiolabeled ␣-factor accumulated in the precursor form, with the remainder rapidly reaching the mature form. However, the translocation block was complete, as the precursor form could not be chased through the translocation pathway. Since DnaJ-related proteins are known to interact with hsp70s and strains containing conditional mutations in a dnaJ-related gene, YDJ1, are defective in translocation of prepro-␣-factor, we looked for a genetic interaction between SSA genes and YDJ1 in vivo. We found that a deletion mutation of YDJ1 was synthetically lethal in a ssa1 ts ssa2 ssa3 ssa4 background. In addition, a strain containing a single functional SSA gene, SSA1, and a deletion of YDJ1 accumulated the precursor form of ␣-factor. However, no genetic interaction was observed between a YDJ1 mutation and mutations in the SSB genes, which encode a second class of cytosolic hsp70 chaperones. These results are consistent with SSA proteins and Ydj1p acting together in the translocation process.