sigma32purification

sigma32purification - C ell Vol 38 383-390 September 1984...

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Cell, Vol. 38, 383-390, September 1984, Copyright 0 1984 by MIT 0092.8674/84/090383-08 $02.00/O The htpR Gene Product of E. coli Is a Sigma Factor for Heat-Shock Promoters Alan D. Grossman, James W. Erickson, and Carol A. Gross Department of Bacteriology University of Wisconsin Madison, Wisconsin 53706 Summary The [email protected] gene of E. coli encodes a positive regulator of the heat-shock response. We have fused the htpR gene to the inducible PL promoter of phage lambda. Overproduction of HtpR following a temperature up- shift resulted in the overexpression of heat-shock proteins. We describe the purification and initial in vitro characterization of the factor controlling ex- pression of heat-shock genes. The factor was the 32 kd hfpR gene product. In vitro, a mixture of HtpR and core RNA polymerase initiated transcription at heat-shock promoters. The sigma factor encoded by rpoD was not required for this reaction. Therefore, HtpR is a sigma factor that promotes transcription initiation at heat-shock promoters. We propose that htpff be renamed rpoH and that the gene product be called sigma-32. Introduction The heat-shock response of E. coli is similar to that of other organisms (Schlesinger et al., 1982). Almost imme- diately after a shift to higher temperature, the rate of synthesis of a class of proteins, the heat-shock proteins (hsps) dramatically increases (Lemaux et al., 1978; Ya- mamori et al., 1978). The function of the heat-shock re- sponse may be similar in procaryotes and eucaryotes since the 70 kd hsp of Drosophila, yeast, and E. coli (DnaK) are homologous (Bardwell and Craig, 1984). In E. coli K12, 17 proteins have been identified as hsps (Neidhardt et al., 1984). Each of these proteins exhibits a characteristic increase in its rate of synthesis upon tem- perature up-shift. In general, when cells are shifted from 30°C to 42°C rates of synthesis increase between 5 and 20 fold. The increased rates of synthesis peak 5-10 min after the up-shift and then decline to new steady-state rates of synthesis somewhat greater than those at the low temperature. The increased rate of synthesis of hsps reflects increased synthesis of their mRNAs (Yamamori and Yura, 1980, 1982; Yamamori et al., 1982) and in- creased transcription initiation at heat-shock promoters (Taylor et al., 1984). The htpR gene maps at 76 min on the E. coli chromo- some (Bachmann, 1983) and is required for a normal heat- shock response. This gene was defined by the htpRl65 mutation, an amber mutation that must be maintained in a strain with a suppressor tRNA. In the presence of a temperature-sensitive (ts) suppressor tRNA the mutant strain is ts for growth (Cooper and Ruettinger, 1975) does not undergo the heat-shock response (Neidhardt and Van Bogelen, 1981; Yamamori and Yura, 1982) and is defec- tive in proteolysis (Baker et al., 1984). The hfpR gene has been cloned (Neidhardt et al., 1983) the gene product identified (Neidhardt et al., 1983; Landick et al., 1984) and the DNA sequence determined (Landick et al., 1984).
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sigma32purification - C ell Vol 38 383-390 September 1984...

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