Effects of rifampicin resistant rpoB mutations on antitermination and interaction with nusA in Escherichia coli

Rifampicin resistant (Rifr mutations map in the rpoB gene encoding the beta subunit of Escherichia coli RNA polymerase. We have used our collection of 17 sequenced Rifr mutations to investigate the involvement of E. coli RNA polymerase in the antitermination systems enhancing expression of delayed early lambda genes or stable RNA. We have found that Rifr mutations affect both lambda N-mediated antitermination and the cellular antitermination system involved in synthesis of stable RNA. Because NusA is involved in antitermination and termination, we also investigated the interaction of NusA and RNA polymerase by determining whether Rifr mutations alter NusA-dependent termination or antitermination in cells with defective nusA alleles. We have shown that Rifr mutations can either enhance or suppress the phenotypes of defective nusA alleles. Most Rifr mutations alter the temperature range over which the nusA1 allele supports lambda N-mediated antitermination. In addition, a number of Rifr alleles restore termination to the nusA10(Cs) and the nusA11(Ts) mutants defective in this process. Our results indicate that the region of the rpoB gene defined by the Rifr mutations is involved in the antitermination process and affects the activity of the NusA protein directly or indirectly.     

Mutant rho factors with increased transcription termination activities. II. Identification and functional dissection of amino acid changes

We have determined the nucleotide sequences of three mutant rho genes encoding hyperfunctional rho proteins (rho S) together with their parent allele, rho-ts702. These mutant rho factors contain the following amino acid changes as deduced from their sequences: (1) the thermo-labile mutant, rho-ts702, has Thr304 substituting for Ala; (2) rho S-77 and rho S-81, which are selectively altered in the primary polynucleotide binding site, share an identical mutation, Leu3----Phe; (3) rho S-82, which is altered in both the primary and secondary polynucleotide binding sites, carries three amino acid substitutions together, Leu3----Phe, Asp156----Asn and Thr323----Ile. Dissection and functional characterization of each mutation in rho S-82 have revealed that Ile323 alone is responsible for alterations in both the secondary RNA interaction and the terminator selectivity observed with the original mutant, rho S-82. Taken together, these results not only confirm our proposal in the accompanying paper that the primary and secondary RNA binding sites differently contribute in determining the overall efficiency and site-specificity of termination, respectively, but also support the possibility that these binding sites exist as structurally distinct domains in rho protein. In contrast, Asn156 was shown to cause decreased termination efficiency, though it had no influence on RNA interactions. Thus, this amino acid residue appears to be associated with still another rate-determining step of termination, for instance, interactions between rho and RNA polymerase. On the basis of Chou-Fasman secondary structure predictions as well as amino acid sequence comparison with F1-ATPase, we discuss how the proposed domains are structurally and functionally related to the putative ATPase reactive center of rho protein.