Escherichia coli rpoC397 encodes a temperature-sensitive C-terminal frameshift in the beta' subunit of RNA polymerase that blocks growth of bacteriophage P2.

Escherichia coli 397c is temperature sensitive for growth at 43.5 degrees C and unable to plate bacteriophage P2 at 33 degrees C. The mutation conferring these phenotypes was mapped to the rpoC gene. RNA synthesis is temperature sensitive in the mutant strain, and the beta' subunit of RNA polymerase isolated from this strain exhibits increased electrophoretic mobility. DNA sequence analysis revealed that the mutation is a deletion of 16 bp, resulting in a frameshift that leads to truncation of the beta' subunit at the carboxy terminus.     

Mapping of a contact for the RNA 3' terminus in the largest subunit of RNA polymerase.

Stalled elongation complexes of Escherichia coli RNA polymerase were prepared carrying the photo-cross-linkable 8-azido derivative of adenine at the 3'-terminus of the nascent RNA chain. Ultraviolet irradiation of such complexes resulted in the cross-linking of radiolabeled RNA exclusively to the beta' subunit of RNA polymerase. The adduct was mapped between Met932 and Trp1020 in the linear sequence of the beta' polypeptide using specific chemical degradation of the cross-linked species.     

Escherichia coli RNA polymerase subunit omega and its N-terminal domain bind full-length beta' to facilitate incorporation into the alpha2beta subassembly.

The omega subunit of Escherichia coli RNA polymerase, consisting of 90 amino acids, is present in stoichiometric amounts per molecule of core RNA polymerase (alpha2betabeta'). The presence of omega is necessary to restore denatured RNA polymerase in vitro to its fully functional form, and, in an omega-less strain of E. coli, GroEL appears to substitute for omega in the maturation of RNA polymerase. The X-ray structure of Thermus aquaticus core RNA polymerase suggests that two regions of omega latch on to beta' at its N-terminus and C-terminus. We show here that omega binds only the intact beta' subunit and not the beta' N-terminal domain or beta' C-terminal domain, implying that omega binding requires both these regions of beta'. We further show that omega can prevent the aggregation of beta' during its renaturation in vitro and that a V8-protease-resistant 52-amino-acid-long N-terminal domain of omega is sufficient for binding and renaturation of beta'. CD and functional assays show that this N-terminal fragment retains the structure of native omega and is able to enhance the reconstitution of core RNA polymerase. Reconstitution of core RNA polymerase from its individual subunits proceeds according to the steps alpha + alpha --> alpha2 + beta --> alpha2beta + beta' --> alpha2betabeta'. It is shown here that omega participates during the last stage of enzyme assembly when beta' associates with the alpha2beta subassembly.     

Temperature-sensitive ribonucleic acid polymerase mutant of Salmonella typhimurium with a defect in the beta' subunit.

Localized mutagenes of Salmonella typhimurium followed by a [3H]uridine enrichment procedure yielded a temperature-sensitive strain with a mutation in the rpo region of the chromosome. Ribonucleic acid (RNA) polymerase (EC 2.7.7.6; nucleoside triphosphate: RNA nucleotidyltransferase) purified from this mutant was considerably less active at the nonpermissive temperature than wild-type enzyme. Furthermore, the enzyme from this mutant, unlike RNA polymerase of previously isolated temperature-sensitive mutants, was as thermostable as wild-type enzyme when preincubated at 50 degrees C. Subunit reconstitution experiments have shown that the temperature sensitivity is caused by an alteration in the beta' subunit of the enzyme.     

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.     

A missense mutation in the rpoC gene affects chromosomal replication control in Escherichia coli.

An RNA polymerase mutant with a single-base-pair change in the rpoC gene affects chromosome initiation control. The mutation, which is recessive, is a G to A transition leading to the substitution of aspartate for glycine at amino acid residue 1033 in the RNA polymerase beta' subunit. The chromosome copy number is increased twofold in the mutant at semipermissive growth temperatures (39 degrees C). In a delta oriC strain, in which chromosome initiation is governed by an F replicon, chromosome copy number is not affected. Plasmid pBR322 copy number is also increased in the mutant at 39 degrees C. The mutation causes a more than fivefold increased expression of the dnaA gene at 39 degrees C. It is conceivable that it is this high DnaA concentration which causes the high chromosome copy number and that the mutant RNA polymerase beta' subunit exerts its effect by altering the expression of the dnaA gene. However, other factors must be affected as well to explain why the RNA polymerase mutant can grow in a balanced fashion with a high chromosome concentration. This is in contrast to wild-type cells, which exhibit higher origin concentrations when DnaA protein is overproduced, but in which the overall DNA concentration is only moderately affected.     

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

Taking advantage of sequence conservation of portions of the alpha, beta, and beta' subunits of RNA polymerase of bacteria and plant chloroplasts, we have designed degenerate oligonucleotides corresponding to these domains and used these synthetic DNA sequences as primers in a polymerase chain reaction to amplify DNA sequences from the chlamydial genome. The polymerase chain reaction products were used as a probe to recover the genomic fragments encoding the beta subunit and the 5' portion of the beta' subunit from a library of cloned murine Chlamydia trachomatis DNA. Similar attempts to recover the alpha subunit were unsuccessful. Sequence analysis demonstrated that the beta subunit of RNA polymerase was located between genes encoding the L7/L12 ribosomal protein and the beta' subunit of RNA polymerase; this organization is reminiscent of the rpoBC operon of Escherichia coli. The C. trachomatis beta subunit overproduced in E. coli was used as an antigen in rabbits to make a polyclonal antibody to this subunit. Although this polyclonal antibody specifically immunoprecipitated the beta subunit from Chlamydia-infected cells, it did not immunoprecipitate core or holoenzyme. Immunoblots with this antibody demonstrated that the beta subunit appeared early in infection.     

A new protein subunit k for RNA polymerase from Xanthomonas campestris pv. oryzae

During the purification of RNA polymerase from Xanthomonas campestris pv. oryzae, a new subunit named k was found to be associated with this enzyme. The removal of subunit k from holoenzyme by DEAE-cellulose column chromatography results in a decrease in specific activity of the enzyme. The readdition of subunit k to subunit k-depleted holoenzyme results in restoration of enzymatic activity. Subunit k increase the activity of RNA polymerase; the activation was in proportion to the concentration of subunit k added. Antiserum against holoenzyme devoid of subunit k was prepared. This antiserum did not react with purified subunit k; therefore, subunit k may not be the proteolytic fragment of the beta, beta', sigma, or alpha subunit. When this antiserum was used to precipitate RNA polymerase obtained from a crude extract of bacterial cells, subunit k was coprecipitated as determined by sodium dodecyl sulfate gel electrophoretic analysis. The molecular mass of subunit k is approximately 29 kDa, and the molar ratio of beta:beta':sigma:alpha:k was estimated to be 1:1:1:2:4. When native Xp10 DNA was used as template, subunit k stimulated subunit k-depleted holoenzyme, but not core enzyme. When the synthetic polynucleotide poly[d(A-T)] was used, subunit k activated both subunit k-depleted holoenzyme and core enzyme. Subunit k also activated the binding of RNA polymerase to template DNA.