Correlation between the 32-kDa sigma factor levels and in vitro expression of Escherichia coli heat shock genes.

Abstract

ABSTIRACT S-30 extracts from Escherichia coli cells were used to express heat shock (HS) and non-HS genes in vitro in a DNA-directed protein synthesis system. The S-30 extracts prepared from cells that have been shifted to 450C express HS genes in vitro =8 times better than extracts from cells at 33TC. In contrast, the expression of non-HS genes in extracts from heat-induced cells is only 40% ofthat seen in extracts from cells at 330C. These results correlate well with the levels of HS ar factor and normal ca factor bound to RNA polymeraswe. Thus, there was an 8-fold increase in the HS or factor and a 60% decrease in the normal or factor associated with RNA polymerase at the higher temperature. Part of the increase in the level of the HS cv factor could be accounted for by a 3-fold increase in the level of HS or factor mRNA during heat induction.There is now considerable information available on the heat shock (HS) response in Escherichia coli. A temperature shift from -30'C to 40-450C results in the increased synthesis of at least 17 proteins (1), and this response is regulated by a 32-kDa protein, the product of the htpR gene (2-4). Based on sequence data that showed that the htpR gene product possessed a high degree of homology with the 70-kDa a-factor (cr70), it was predicted (5) that this protein functioned as a oa factor (o&32). This has been clearly demonstrated by Grossman et al. (6) who showed that the HS RNA polymerase (Eo-32) could recognize specific HS sequences present on HS genes (7). Our laboratory has demonstrated that the htpR gene on a plasmid template could be expressed in vitro by the normal cellular RNA polymerase (Eo-70) to yield &2, which was purified in the form of Eo2 (8). In addition, it was shown that the Eo-32 produced in vitro (and in vivo) preferentially stimulated the expression of HS genes such as dnaK and groEL.A major unanswered question relative to the HS response is how heat alters the level or activity of &r32 in cells. The present studies were initiated to determine whether changes in &32 levels during HS correlated with the expression of HS genes in vitro. MATERIALS AND METHODSPlasmids and E. coli Strains. Plasmid pKT200, which contains groEL, and plasmid pCG203, which contains the dnaK gene, were kindly supplied by C. Georgopoulos (University of Utah). Plasmid pFN97 containing the a-32 gene (4) was kindly supplied by F. Neidhardt (University of Michigan). Purified plasmid DNAs were obtained from cleared lysates after two successive centrifugations through CsCl/ ethidium bromide. A .32 mutant strain of E. coli (JC7623-htpR::TnS) was constructed in the laboratory of Graham C. Walker (Massachusetts Institute of Technology) and kindly supplied to us by F. Neidhardt. E. coli B cells were grown in 2 liters of LB (Luria-Bertani) medium (9) in an airlift fermenter (Bethesda Research Laboratories). Cells were grown to midlogarithmic phase at 330C and shifted first to 40'C and then to 450C. Each shift took -4 min, and once the desired temperature was reached, the cells were immedia...

How a mutation in the gene encoding sigma 70 suppresses the defective heat shock response caused by a mutation in the gene encoding sigma 32

“…In wild-type cells, the transient increase in heat shock protein expression is mediated by a transient increase in the intracellular concentration of &2 (17,26,27 30'C or at various times after a shift to 42°C, immunoprecipitated with anti-GroEL antibody as described in Materials and Methods, and separated by SDS-polyacrylamide gel electrophoresis. The gels were dried and subjected to autoradiography.…”

“…The transient increase in expression of heat shock genes after temperature upshift results from increased transcription initiation at heat shock promoters (6,28,31,32,33), which is mediated by a transient 20-fold increase in the amount of &32 per cell (17,26,27 …”

The activity of sigma 32 is reduced under conditions of excess heat shock protein production in Escherichia coli.

“…Changes in the concentration of this alternate a-factor are responsible for regulating transcription of heat shock genes during heat shock. Following temperature upshift, the intracellular level of o^^ increases transiently, leading to a burst of heat shock gene transcription (Lesley et al 1987;Skelly et al 1987;Straus et al 1987). Artificial overproduction of o^^ without a temperature shift also results in the induction of heat shock proteins (Grossman et al 1987).…”

Heat shock protein GroE of Escherichia coli: key protective roles against thermal stress.

“…to rapid and transient increase in the o^^ level (Grossman et al 1987;Skelly et al 1987;Straus et al 1987).…”