RpoB8, a rifampicin-resistant termination-proficient RNA polymerase, has an increased Km for purine nucleotides during transcription elongation

Abstract

The rpoB8 allele of Escherichia coli maps to the beta-subunit of RNA polymerase and confers rifampicin resistance as well as increased termination at both intrinsic and rho-dependent terminators in vivo. This phenotype suggests that the mutant is defective in an enzymatic property of RNA polymerase important for all termination events. We analyzed the in vitro transcription properties of this enzyme to determine the nature of the defect. As compared with the wild-type enzyme, RpoB8 exhibits enhanced pausing and a significant reduction in rate of elongation on natural templates. In addition, RpoB8 RNA polymerase has a 3-5-fold higher Km for purine nucleotides during elongation on synthetic templates. In contrast, both the mutant and wild-type enzyme have the same initiation Km for ATP. Kinetic analysis indicates that RpoB8 is likely to be defective in nucleotide binding during elongation, suggesting that the mutational alteration affects the binding site. We show that our data are consistent with the idea that the altered Km underlies the altered pausing and elongation properties of the enzyme, and we discuss the implication of these results for the termination proficiency of the mutant strain.