Mutation slowing down the degradation of the beta beta'-subunits of Escherichia coli RNA-polymerase

The rpoC1 ts mutation affecting the RNA polymerase beta' subunit accelerates synthesis of RNA polymerase beta beta' subunits at 42 degrees C, while the surplus amount of subunits degrades in an hour's time. In a Ts strain with two RNA polymerase mutations, rpoC1 and rpoB251, we obtained a ts+ reversion designated opr24 which slows down degradation of surplus beta beta' subunits. The slowing down of degradation and the resulting accumulation of beta beta' subunits does not affect the kinetics of beta beta' subunit synthesis after the transfer to 42 degrees C. The effects of the opr24 are allele non-specific. The mutation also slows down degradation of beta' subunit and the amber fragment of beta subunit in the strain with subunit amber mutation rpoB22. Besides, the opr24 mutation reduces proteolysis of anomalous proteins containing canavanine. The opr24 mutation has been mapped between 17 and 21 minutes on the Escherichia coli map.     

Functionally important site in the vicinity of the amino-terminus of the Escherichia coli RNA polymerase beta subunit

We have analyzed the interaction of monoclonal antibodies against Escherichia coli RNA polymerase with products of its limited proteolysis. Two major proteolytic fragments of molecular masses 107 and 43 kDa originate as a result of a single cleavage in the vicinity of the 980th amino acid residue. Anti-beta subunit monoclonal antibody PYN-2 inhibiting RNA polymerase activity at the stage of RNA elongation reacts with an epitope located between the amino-terminus and the 50th amino acid residue of the beta subunit. DNA sequencing has shown that the RNA polymerase mutation rpoB22 converts the Gln(1111) codon of the beta subunit gene into the amber codon. An epitope for the monoclonal antibody PYN-6 was located between the major site of proteolytic cleavage and Gln(1111) of the beta subunit.     

Mutations in the Escherichia coli RNA-polymerase beta-subunit gene cloned in a multicopy plasmid

A multicopy plasmid pLMN1 expressing a wild type rpoB gene encoding Escherichia coli RNA polymerase beta subunit gene was constructed. Introduction of this plasmid into rifampicin-resistant RpoB mutants makes them rifampicin-sensitive. Rifampicin-resistant clones appear in such strains with frequencies up to 10(-3), due to recombinational (recA-dependent) transfer of rif-r mutations from chromosome to pLMN1. This provides a simple selection procedure for transfer of any rpoB mutation, together with a rif-r mutation from a chromosome to pLMN1. In this way, we transferred rpoB22 amber mutation to pLMN1 for localization of the mutant codon by DNA sequencing.     

Autogenous regulation of RNA polymerase beta subunit synthesis in vitro.

The effects of Escherichia coli RNA polymerase and its subassemblies and subunits on the in vitro synthesis of beta subunit directed by DNA from a lambda transducing phage lambdadrif+-6 were investigated. This phage carries the structural gene (rpoB) for beta subunit as well as the genes for EF (translation elongation factor)-Tu, some ribosomal proteins, and stable RNAs of the E. coli chromosome. Among the RNA polymerase proteins examined, the two oligomers, holoenzyme and alpha2beta complex, repressed the synthesis of only the beta subunit but not of other proteins encoded by the phage DNA. The results indicate that the expression of at least the betabeta' (rpoBC) operon is under autogenous regulation, in which both holoenzyme and alpha2beta complex function as regulatory molecules with repressor activity.     

Overexpression and purification of a biologically active rifampicin-resistant beta subunit of Escherichia coli RNA polymerase

The gene rpoB (rifD 18), which encodes rifampicin-resistant beta subunit of Escherichia coli RNA polymerase, has been placed on an overexpression plasmid under the control of bacteriophage T7 promoter. Induction of the T7 RNA polymerase gene in the host cells resulted in extensive overproduction of the beta polypeptide. Most of the overproduced material was recovered from cell lysates in insoluble form and was solubilized by extraction with 6 M urea. Purified overproduced beta subunit was added, in molar excess, to urea-denatured rifampicin-sensitive RNA polymerase. Upon removal of urea by dialysis, the reconstituted enzyme became rifampicin-resistant, indicating that overproduced beta subunit can be efficiently assembled into functional holoenzyme.     

Molecular characterization of the rpoB gene in Brucella species: new potential molecular markers for genotyping

The rpoB gene encoding the beta subunit of the DNA-dependent RNA polymerase was molecularly characterized by PCR amplification and DNA sequencing in 26 Brucella reference strains by using primers selected according to the B. melitensis 16 M rpoB published sequence. Comparison of the rpoB nucleotide sequence of all Brucella strains analysed revealed specific nucleotide variations associated with different Brucella species and biovars. 17 rpoB alleles were recognized and new Brucella typing is proposed. Our results suggest that the rpoB gene polymorphism can be used to identify all Brucella species and most of the biovars, offering an improvement over conventional typing methods.     

The role of the beta-subunit of RNA-polymerase in specific recognition of promoters

The complex formation of T7 DNA with RNA polymerase from E. coli B/r WU-36-10-11-12 (E. coli W12) and its rifampicin resistant mutant with highly pleiotropic effect--rpoB409 was studied. As shown earlier rpoB409 RNA polymerase differs from the normal enzyme by the selection of RNA synthesis from early promoters of DNA from T7 and T4 phages. The change in the RNA specificity synthesis due to rpoB409 mutation was shown to occur at the stage of RNA polymerase interaction with DNA before open promoter complex formation. The data obtained together with the fact of highly pleiotropic effect of the rpoB409 mutation indicate that RNA polymerase beta-subunit takes part in specific recognition of promoters.     

rpoB mutation in Escherichia coli alters control of ribosome synthesis by guanosine tetraphosphate.

An isogenic pair of relA+ and relA strains of Escherichia coli B/r with a mutation in the RNA polymerase subunit gene rpoB (Rifr) was isolated in which the relationship between guanosine tetraphosphate (ppGpp) concentration and stable RNA (rRNA, tRNA) gene activity was altered. The RNA polymerase in the rpoB strains was found to be about 20-fold more sensitive to ppGpp with respect to its stable RNA promoter activity than was the wild-type enzyme. The existence of such mutants is consistent with the idea that ppGpp interacts with the RNA polymerase enzyme and thereby alters its promoter selectivity, i.e., reduces its affinity for the stable RNA promoters. Under most conditions, the rpoB mutants had a reduced rate of growth and about a 10-fold-reduced intracellular concentration of ppGpp compared with the rpoB wild-type strains. The reduction of the level of ppGpp in the rpoB mutants during exponential growth was presumably a reflection of an indirect effect of the rpoB mutation on the control of relA-independent ppGpp metabolism.     

The spectrum of spontaneous rifampin resistance mutations in the rpoB gene of Bacillus subtilis 168 spores differs from that of vegetative cells and resembles that of Mycobacterium tuberculosis

Mutations causing rifampin resistance in vegetative cells of Bacillus subtilis 168 have thus far been mapped to a rather restricted set of alterations at either Q469 or H482 within cluster I of the rpoB gene encoding the beta subunit of RNA polymerase. In this study, we demonstrated that spores of B. subtilis 168 exhibit a spectrum of spontaneous rifampin resistance mutations distinct from that of vegetative cells. In addition to the rpoB mutations Q469K, Q469R, and H482Y previously characterized in vegetative cells, we isolated a new mutation of rpoB, H482R, from vegetative cells. Additional new rifampin resistance mutations arising from spores were detected at A478N and most frequently at S487L. The S487L change is the predominant change found in rpoB mutations sequenced from rifampin-resistant clinical isolates of Mycobacterium tuberculosis. The observations are discussed in terms of the underlying differences of the DNA environment within dormant cells and vegetatively growing cells.     

Genes that protect against the host-killing activity of the E3 protein of Bacillus subtilis bacteriophage SPO1.

A cloned rpoB gene, specifying an apparently mutant RNA polymerase beta subunit, protected Escherichia coli against the cytocidal effects of the E3 protein of bacteriophage SPO1, suggesting that RNA polymerase is the primary cellular target of the E3 protein. Two segments of the wild-type E. coli genome, one of which specifies a suppressor of dnaK mutations, and thus, possibly, a molecular chaperone, also provided protection when overexpressed, but wild-type rpoB did not.