Expression of cloned rpoB gene of Escherichia coli: a genetic system for the isolation of dominant negative mutations and overproduction of defective beta subunit of RNA polymerase

Abstract

The rifampin resistance rifD18 allele of rpoB, carried on the expression plasmid pXT71, is controlled by a strong bacteriophage T7 late promoter and two weak Escherichia coli promoters. Depending on the host strain, pXT71 specifies different levels of Rifr ,1 subunit, providing a system for the isolation, maintenance, and overexpression of dominant lethal alleles of rpoB. In rpoB+ hosts, pXT713 confers the RiF phenotype on the Rif' host. Negative rpoB mutations in the plasmid DNA can thus be scored by screening transformants for Rif. In an rpoB(Am) supD(Ts) host in which chromosomal rpoB expression is decreased as the temperature goes up, some of the negative plasmid-borne rpoB mutations displayed a dominant phenotype. In a host harboring inducible T7 RNA polymerase, the defective subunits could be overexpressed independently of the E. coli transcriptional machinery. With this system, we isolated several negative rpoB mutations induced in vitro by hydroxylamine. Seven of the mutant rpoB alleles, when overexpressed, were found to specify normal-size polypeptides. Two of them displayed the dominant lethal phenotype in the rpoB(Am) supD(Ts) background. We also constructed a mutation (rpoB1800) in which 24 carboxy-terminal amino acids were substituted with a random 19-amino-acid sequence. The nonfunctional rpoB1800 polypeptide was isolated and assembled in vitro into the core enzyme molecule.The DNA-dependent RNA polymerase (RNAP) of Escherichia coli consists of four essential subunits, ax, 1, 1', and a, encoded by the genes rpoA, rpoB, rpoC, and rpoD, respectively (7,8,27,47). The assembled enzyme performs a variety of biochemical functions (30, 44) and interacts with numerous regulatory factors (40,42). To dissect these functions genetically, one would like to obtain mutations of rpo genes resulting in a fully assembled RNAP with changed properties. Since RNAP is an essential enzyme, this approach has so far been mostly limited to viable mutants, e.g., rifampin-resistant alleles of rpoB (see reference 22 and references cited therein) or permissible substitutions resulting from rpoB nonsense suppression (14,33,35).Amino acid substitution mutations affecting individual essential functions of RNAP are expected to be dominantly lethal (19), at least under conditions in which the mutant allele is overexpressed relative to the wild-type allele. To obtain such mutants, an experimental system is needed that would meet the following requirements. First, mutations should be induced in a merodiploid, in an underexpressed copy of the gene, to make sure that the mutant product will not poison the cell by "diluting" the wild-type subunit during RNAP assembly. Second, the gene under study should nevertheless be expressible to a certain level so that the mutants sought can be identified by a phenotype change. Third, there should be a way to overexpress the subunit in question on a large scale and independently of E. coli RNAP so that the mutant product can be obtained for biochemical study. Our objective is to develop such a syst...

A beta subunit mutation disrupting the catalytic function of Escherichia coli RNA polymerase.

“…The mutation rpoBl855 was originally isolated as a dominant lethal mutation, after random mutagenesis of the expression plasmid pXT7,3, which carries a Rif-resistant allele of rpoB (16). To map rpoB1855, a series of in vitro exchanges of DNA fragments (Fl, F2, F3, and F4) was performed between plasmids as illustrated in Fig.…”

“…The plasmids pXT7,3, its rpoB1855-carrying derivative pJL97 (16), and pMKA92 (14) were described. pMKA351, pMKA335, and pMKA312 were isolated in the course of linker-insertion mutagenesis of pMKA92 (M.K., unpublished observations).…”

Cloning and characterization of RNA polymerase core subunits of Chlamydia trachomatis by using the polymerase chain reaction

“…Cell lysis conditions were selected to minimize the amount of detergent in an effort to prevent dissociation or denaturation of the multisubunit enzyme (10% glycerol and 0.2% Triton X-100; see Materials and Methods). Figure 5 illustrates E. coli 13' C. trach 13' E. coli 13 (Fig. 5, lanes 9 and 6) that was absent from uninfected cells (Fig.…”

“…These two proteins were 51% identical over the 122 amino acid residues (data not shown). Second, downstream of the 13 subunit gene was found the gene encoding the 13' subunit homolog. The amino acid sequence surrounding the XbaI site showed 48% identity over a 116-amino-acid stretch with the E. coli 1' subunit, strongly suggesting that this gene does encode the 13' subunit of C. trachomatis RNA polymerase (Fig.…”

“…The material was electrophoresed on a preparative 12% SDSpolyacrylamide gel (12). A strip from the gel was stained briefly in 10% acetic acid containing 0.25% Coomassie blue R250, and the band corresponding to the overexpressed 13 protein fragment was excised. This excised gel fragment was VOL.…”