In E. coli, σ⁷⁰ is the major but not the only sigma factor. Several other sigma factors direct RNA polymerase to bind to different sets of promoters. The second sigma factor to be discovered was σ³², which participates in the expression of genes involved in the heat shock response. When the environmental temperature suddenly rises, cellular production of about 20 proteins increases. These proteins help protect the cell from any ill effects of the higher temperature. Some of the proteins are chaperones that facilitate protein folding (see Chapter 4). In the 1970s, a gene required for normal heat shock response was discovered and named htpR. Carol Gross and her colleagues later identified its protein product, HtpR, as the first alternative sigma factor in E. coli (several had previously been discovered in B. subtilis); they renamed the gene rpoH (rpo genes encode RNA polymerase subunits) and named the protein σ³². Was this renaming justified?

Gross and coworkers purified the 32 kDa HtpR protein. At a late stage in the purification, they ran the protein preparation over a cation-exchange column. For each fraction they collected, they ran a sample on a polyacrylamide gel (Figure 1a) and assayed the fraction for RNA synthesis directed by a heat shock promoter called P_HS; the RNA product of this assay was run on a separate polyacrylamide gel (Figure 1b). Fraction numbers are shown above the gel lanes. (The M lane has molecular weight markers; the H lane has purified RNA polymerase; nt indicates number of nucleotides.)

The investigators then reconstituted the RNA polymerase, using purified RNA polymerase subunits but replacing σ⁷⁰ with the 32 kDa protein that was now fairly pure. They used σ⁷⁰-containing RNA polymerase as a control. To find out whether the 32 kDa protein could direct RNA synthesis from more than one heat shock promoter, they constructed a plasmid with two such promoters (P1 and P2), then cut it with restriction enzymes. Transcription from the promoters would generate RNA transcripts with lengths of 215 and 140 nucleotides, respectively. The results are shown in Figure 2. Lanes 1 and 3 show RNA synthesis by the polymerase with the 32 kDa protein; lanes 2 and 4 show synthesis by the polymerase with σ⁷⁰.

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