Sequence analysis of two temperature-sensitive mutations in the alpha subunit gene (rpoA) of Escherichia coli RNA polymerase

The rpoA gene of Escherichia coli encodes the α subunit of the DNA-dependent RNA polymerase. Two mutant alleles, rpoA101 and rpoA112, both of which produce RNA polymerase with altered thermostability and reduced fidelity of transcription in vitro (Ishihama et al. (1980) J. Mol. Blol. 137, 137-150), have been analyzed in details. The mutations were found to be responsible for the temperature-sensitive growth by complementation test using a rpoA-expression plasmld. Each mutant allele was amplified from total cell DNA by PCR (polymerase chain reaction) and directly sequenced. Both the mutant rpoA genes were found to carry a single base transition which leads to a substitution of Cys for Arg at the position 191 (rpoA101) or 45 (rpoA112), respectively. Since the rpoA112 mutation causes the defect in RNA polymerase assembly (Kawakami & Ishihama (1980) Biochemistry 19, 3491-3495), the amino-terminal region of α including the position 45 was considered to play an important role in subunit assembly.