The Escherichia coli transcription factor sigma 32 binds to core RNA polymerase to form the holoenzyme responsible for transcription initiation at heat shock promoters, utilized upon exposure of the cell to higher temperatures. We have developed two ways to assay sigma 32-dependent RNA synthesis in E. coli. The plasmid-borne reporter gene for both is lacZ (beta-galactosidase), driven by the groE promoter. In one application, the cells are exposed to a temperature of 42 degrees C in order to induce accumulation of endogenous sigma 32. The other involves isopropylthiogalactopyranoside (IPTG)-induced synthesis of sigma 32 at 30 degrees C from a gene contained on a second plasmid. The latter employs DnaK(-) cells, which additionally contained a second mutation, inactivating the endogenous sigma 32 gene (Bukau and Walker, EMBO J. 9:4027-4036, 1990). These assays were used to delineate the sequences CTTGA (-37 to -33) and GNCCCCATNT (-18 to -9) as important for sigma 32 promoter activity. At each of the specified base pairs, substitutions were found which reduced promoter activity by greater than 75%. Activity was also dependent upon the number of base pairs separating the two regions.