Abstract
Among the earliest rpoBC mutations identified are three suppressors of the conditional lethal rho allele, rho201. These three mutations are of particular interest because, unlike rpoB8, they do not increase termination at all rho-dependent and rho-independent terminators. rpoB211 and rpoB212 both change Asn-1072 to His in conserved region H of rpoB (betaN1072H), whereas rpoC214 changes Arg-352 to Cys in conserved region C of rpoC (beta'R352C). Both substitutions significantly reduce the overall rate of transcript elongation in vitro relative to wild-type RNA polymerase; however, they probably slow elongation for different reasons. The nucleotide triphosphate concentrations required at the T7 A1 promoter for both abortive trinucleotide synthesis and for promoter escape are much greater for betaN1072H. In contrast, beta'R352C and two adjacent substitutions (beta'G351S and beta'S350F), but not betaN1072H, formed open complexes of greatly reduced stability. The sequence in this region of beta' modestly resembles a region of Escherichia coli DNA polymerase I that contacts the phosphate backbone of DNA in co-crystals. Core determinants affecting open complex formation do not reside exclusively in beta', however, since the Rifr mutation rpoB2 in beta also dramatically destabilized open complexes. We suggest that the principal defects of the two Rho-suppressing substitutions may differ, perhaps reflecting a greater role of beta region H in nucleoside triphosphate-binding and nucleotide addition and of beta' region C in contacts to the DNA strands that could be important for translocation. Although both probably suppress rho201 by slowing RNA chain elongation, these differences may lead to terminator specificity that depends on the rate-limiting step at different sites.
Highlights
We suggest that the Most progress in identifying functionally important regions of principal defects of the two Rho-suppressing substitutions may differ, perhaps reflecting a greater role of  region H in nucleoside triphosphate-binding and nucleotide addition and of  region C in contacts to the DNA strands that could be important for translocation
More recently we reported several clusters of substitutions in conserved regions of Ј that alter pausing and termination (Weilbaecher et al, 1994), streptolydigin-resistance was found to result from amino acid substitutions in one of these clusters that corresponds to the amanitin-resistance region of the largest subunit of RNA polymerase II (Severinov et al, 1995), and conserved aspartic acid residues that appear to chelate activesite Mg2ϩ ions were identified using the Ј homolog in yeast RNA polymerase III (Dieci et al, 1995)
N1072H and ЈR352C Increased the Apparent Km for both Priming and Substrate Nucleotides during Initiation—In principle, the defects in elongation we found could either reflect a fundamental catalytic defect, which should be evident at all nucleotide addition events, or might be specific to the elongation phase of transcription, such as would be the case if they affected movement of the DNA through the enzyme
Summary
Unlike the rpoB8(Rif r) mutant that was isolated as a suppressor of rho201 (Guarente and Beckwith, 1978) as well as independently by several other criteria (reviewed in Jin and Gross (1988)), these mutations do not cause general defects in termination, but rather alter termination only subtly at a few -dependent and -independent terminators (Jin and Gross, 1989) To continue this investigation, we identified the precise amino acid substitutions in rpoB211, rpoB212, and rpoC214, finding that both rpoB211 and rpoB212 specify N1072H and that rpoC214 specifies ЈR352C, and purified both mutant RNA polymerases. ϨpJH76 CAG 14179rpoB211 CAG 14179rpoC214 W3110 rpoC::S. aureus protein A, trpR tnaA2 zja::kan ⌬(recA-srl)306 srl-301::Tn10 RL676 pRW308(ЈS350F) RL676 pRW308(ЈG351D)
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
