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...

A polyclonal antibody to a'32, the heat shock or factor, has been used to show the presence oflow levels of ar 32 in Escherichia coli RNA polymerase preparations (Ea'70), which explains the observed in vitro activity of Ea'70 towards heat shock genes. The a 32 antibody cross-reacts with DnaK, and DnaK has been found associated with purified preparations of both Ea'70 and the heat shock RNA polymerase, Ear 32.In Escherichia coli at least 17 proteins have been identified as members of the heat shock (HS) regulon (1). When E. coli cells growing at 30'C are shifted to 420C, the rates of synthesis of HS proteins increase 5-to 20-fold, reaching a peak within 5 to 15 min after the temperature shift. Thereafter, synthesis declines until a new rate characteristic of the higher temperature is attained.It is now well established that the HS response in E. coli requires the presence of the htpR gene product, a 32-kDa protein (2-4). This protein functions as a ar factor (cr32) by binding to core DNA-directed RNA polymerase (E; EC 2.7.7.6) (5, 6), to form a HS RNA polymerase (Ecr32) that can recognize specific promoter sequences on HS genes (7). Eo-32, relatively free of Eor70 [RNA polymerase containing the normal 70-kDa a-factor (cr70)], has been purified from E.