Amino Acid Substitutions in the Two Largest Subunits of RNA Polymerase That Suppress a Defective Rho Termination Factor Affect Different Parts of the Transcription Complex

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 -dependent and -independent terminators. rpoB211 and rpoB212 both change Asn-1072 to His in conserved region H of rpoB (␤N1072H), whereas rpoC214 changes Arg-352 to Cys in conserved region C of rpoC (␤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 ␤N1072H. In contrast, ␤R352C and two adjacent substitutions (␤G351S and ␤S350F), but not ␤N1072H, formed open complexes of greatly reduced stability. The sequence in this region of ␤ 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 ␤, however, since the Rif r mutation rpoB2 in ␤ 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 ␤ 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. 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.The two largest subunits of Escherichia coli RNA polymerase, ␤Ј (M r 155,063) and ␤ (M r 150,538) are homologous to the largest and second largest subunits, respectively, of all multisubunit RNA polymerases (Allison et al., 1985; Briggs et al., 1985;Falkenberg et al., 1987;Hudson et al., 1988;Leffers et al., 1989;Patel and Pickup, 1989). Most catalytic and regulatory functions of RNA polymerase appear to involve these subunits. Thus ␤ and ␤Ј are likely to form an enzymatic platform for RNA synthesis common to all forms of life, making elucidation of structure/function relationships in these subunits in a well studied prokaryote an ideal approach to understanding the mechanism of transcription. Sequence comparisons and recent crystallographic studies of single subunit DNA and RNA polymerases reveal broadly similar catalytic regions for both classes of enzyme (Steitz et al

The rpoB mutants destabilizing initiation complexes at stringently controlled promoters behave like “stringent” RNA polymerases in Escherichia coli

“…The complexes formed between RNAP and stringent promoters on linear DNA template are extremely unstable and their half-lives last only seconds, the least-stable complexes in E. coli known to us at the present time. [For example, the half-life of initiation complexes of a nonstringent strong promoter, T7A1, is about 30 min on linear DNA template (22), a value regarded as relatively unstable compared with other nonstringent promoters in E. coli.]…”

Fitness Cost of Rifampin Resistance in Neisseria meningitidis: In Vitro Study of Mechanisms Associated with rpoB H553Y Mutation

“…Upregulation of greA may be associated with the stringent phenotype of the H553Y mutant, as there is evidence that GreA plays a major role in the ppGpp-DksA regulatory network (66). nusA upregulation is also expected because equivalent mutations in E. coli (RpoB H526Y) and Bacillus subtilis (H482Y) are responsible for severe defects in transcription termination, and the E. coli RpoB H526Y substitution suppresses the antitermination defect of the nusA1 allele (67)(68)(69).…”

BglG, the transcriptional antiterminator of the bgl system, interacts with the β′ subunit of the Escherichia coli RNA polymerase

“…Second, substitutions in homologous segments in ␤Ј and in the eukaryotic RNA polymerase II largest subunit, which is a homologue of ␤Ј, were shown to confer resistance to antibiotics that block transcription elongation [streptolydigin and ␣-amanitin, respectively (47,48)]. Third, a mutation in ␤Ј was shown to suppress the rho201, allele which produces a defective Rho termination factor (49,50). Fourth, mutations in rpoC, which block Nun-dependent termination at nutR have been isolated (51).…”