Non-coordinate synthesis of RNA polymerase beta beta'' subunits in a temperature-sensitive beta''-subunit mutant of Escherichia coli

Summary On exposure to high temperature of a temperature-sensitive RNA polymerase subunit (rpoC92) mutant of Escherichia coli, selective reduction was observed in the rate of synthesis of a group of proteins including RNA polymerase subunit. The finding that the synthesis of subunit but not subunit was specifically repressed in this mutant grown at non-permissive temperature indicates that the functionally intact RNA polymerase is required for the synthesis of subunits be coordinated. In addition, the assembly of newly synthesized RNA polymerase subunits was inefficient in this mutant at the steps where altered subunit was involved, and the unassembled enzyme subunits were rapidly and preferentially degraded. During recovery to non-restricted growth, the synthesis of both and subunits was transiently enhanced in parallel leading to recovery of the intracellular concentration of functional RNA polymerase.     

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.     

An amber mutation in the gene encoding the beta'' subunit of Escherichia coli RNA polymerase.

An Escherichia coli strain carrying an amber mutation (UAG) in rpoC, the gene encoding the beta prime subunit of RNA polymerase, was isolated after mutagenesis with nitrosoguanidine. The mutation was moved into an unmutagenized strain carrying the supD43,74 allele, which encodes a temperature-sensitive su1 amber suppressor, and sue alleles, which enhance the efficiency of the suppressor. In this background, beta prime is not synthesized at high temperature. Suppression of the mutation by the non-temperature-sensitive amber suppressor su1+ yields a protein which is functional at all temperatures examined (30, 37, and 42 degrees C).     

Regulated synthesis of RNA polymerase II polypeptides in Chinese hamster ovary cell lines.

RNA polymerase II polypeptides present in [35S]methionine-labeled Chinese hamster ovary (CHO) cell extracts have been quantitatively immunoprecipitated with an anti-calf thymus RNA polymerase II serum. Analyses of the immunoprecipitates on sodium dodecyl sulfate polyacrylamide gels indicated that the immunoprecipitated polymerase II of both wild type CHO cells and the alpha-amanitin-resistant mutant Ama1 had polypeptides of molecular weight 214,000, 140,000, 34,000, 25,000, 23,000, 20,500, and 16,500. In heterozygous alpha-amanitin-resistant/alpha-amanitin-sensitive hybrid CHO cells, growth in the presence of alpha-amanitin results in the inactivation of the alpha-amanitin-sensitive RNA polymerase II activity and a compensating increase in the activity of the alpha-amanitin-resistant enzyme. Determination of the rates of synthesis and degradation of RNA polymerase II polypeptides using [35S]methionine labeling and polymerase II immunoprecipitation demonstrated that this increase in activity of alpha-amanitin-resistant polymerase II resulted from a co-ordinate increase in the rate of synthesis of at least three polypeptides of RNA polymerase II. At the same time, there was an enhanced rate of degradation of the alpha-amanitin-inactivated RNA polymerase II polypeptides.     

Peptide mapping of Bacillus subtilis RNA polymerase alpha factors and core-associated polypeptides

An analysis of the peptide maps of the sigma factors and core-associated subunits of Bacillus subtilis RNA polymerase has revealed that all the sigma factors ad core-associated polypeptides are derived from separate genes and are not proteolytically modified products of the major 55,000-dalton sigma factor. A comparison of the peptide pattern of the major B. subtilis and Escherichia coli sigma factors revealed limited homology between them. Furthermore, antibody prepared against the 55,000-dalton B. subtilis sigma factor cross-reacted against the E. coli sigma factor, but not against any of the other B. subtilis sigma factors and core-associated polypeptides. These results unambiguously demonstrate the independently derived nature of the B. subtilis RNA polymerase core-associated subunits and the partial relationship between the major sigma factors of B. subtilis and E. coli.     

The influence of chloramphenicol on synthesis of ribosomes and beta and beta' subunits of RNA polymerase]

The effect of low chloramphenicol concentrations on the biosynthesis of RNA, ribosomal proteins and RNA polymerase in E. coli CP 78 cells was studied. When protein synthesis was decreased by 50--70%, 14C-uracil incorporation in DNA increased twice, the rRNA synthesis being stimulated preferentially. In the presence of antibiotic the RNA/DNA ratio increased from 5,7 to 13,3. The differential rate of r-protein synthesis increased simultaneously with the stimulation of rRNA synthesis, so that alphar rises from 0,083 (without antibiotic) to 0,122 and 0,161 at 5 and 10 microgram/ml of chloramphenicol, respectively. The inhibition of protein synthesis by chloramphenicol is accompanied also by the increase of differential rate of synthesis of beta and beta' subunits of RNA polymerase. In the presence of 5 and 10 microgram/ml of chloramphenicol, alphap increased from 0,90% to 1,44 and 1,57%, respectively. It is assumed that the genes for beta and beta' subunits of RNA polymerase as the ribosomal genes are negatively controlled by guanosine tetraphosphate which intracellular concentration decreased in the presence of chloramphenicol. The known data on the influence of streptolydigin and rifampicin on the RNA polymerase biosynthesis are discussed in view of proposed hypothesis.     

Glucocorticoids modify the rate of ribosomal RNA synthesis in rat thymus cells by regulating the polymerase elongation rate

The mechanism by which glucocorticoids inhibit RNA polymerase A activity, and hence rRNA synthesis, in rat thymus cells has been investigated. Studies of the intranuclear distribution of RNA polymerase A between chromatin bound ("engaged") and unbound ("free") forms revealed that the steroid-mediated inhibition of the activity of the "engaged" form of the enzyme was not accompanied by significant changes in "free" pool activity. In the presence of rifamycin AF/0-13, an inhibitor of re-initiation of RNA polymerase A, the rate of [3H]UMP incorporation into RNA was slower in nuclei from steroid-treated cells than in those from control cells, although in both conditions similar plateau levels of UMP incorporation were attained. Direct measurements of the numbers of transcribing RNA polymerase A molecules and of elongation rates showed that the inhibition of pre-rRNA synthesis was the result of a decrease in enzyme elongation rate; no significant change was observed in the number of transcribing enzymes. The steroid-induced inhibition of pre-rRNA synthesis was selectively abolished by mild proteolysis of nuclei, suggesting the involvement of a labile, regulatory glucocorticoid-induced protein. It is concluded that glucocorticoid treatment of rat thymus cells decreases 45S rRNA synthesis primarily by decreasing the polyribonucleotide elongation rate of RNA polymerase A, possibly by modification of the enzyme.     

Identification of an amber fragment of the beta subunit of Escherichia coli RNA polymerase: a yardstick for measuring controls on RNA polymerase subunit synthesis.

Summary An amber fragment of the subunit of Escherichia coli RNA polymerase has been recovered from strains carrying the rpoB12 amber mutation, indicating that the B12 mutation resides in the structural gene for the subunit. The fragment is readily assayed and can be used to determine the degree of expression of a single rpoB cistron in strains haploid or diploid for this region. These studies confirm that the bacterial mechanism, which can compensate for reduced translation of the message, operates by the co-ordinate induction of rpoB and rpoC. Furthermore, I show that rpo control depends upon cistron(s) located on the F factor, KLF10, whose product(s) can act negatively in trans on rpoBC expression.     

RNA aptamers selected against DNA polymerase beta inhibit the polymerase activities of DNA polymerases beta and kappa

DNA polymerase β (polβ), a member of the X family of DNA polymerases, is the major polymerase in the base excision repair pathway. Using in vitro selection, we obtained RNA aptamers for polβ from a variable pool of 8 × 10¹² individual RNA sequences containing 30 random nucleotides. A total of 60 individual clones selected after seven rounds were screened for the ability to inhibit polβ activity. All of the inhibitory aptamers analyzed have a predicted tri-lobed structure. Gel mobility shift assays demonstrate that the aptamers can displace the DNA substrate from the polβ active site. Inhibition by the aptamers is not polymerase specific; inhibitors of polβ also inhibited DNA polymerase κ, a Y-family DNA polymerase. However, the RNA aptamers did not inhibit the Klenow fragment of DNA polymerase I and only had a minor effect on RB69 DNA polymerase activity. Polβ and κ, despite sharing little sequence similarity and belonging to different DNA polymerase families, have similarly open active sites and relatively few interactions with their DNA substrates. This may allow the aptamers to bind and inhibit polymerase activity. RNA aptamers with inhibitory properties may be useful in modulating DNA polymerase actvity in cells.     

Necessity of polyamines for maximum in vivo synthesis of beta beta' subunits of RNA polymerase

The possibility that polyamines can stimulate the in vivo synthesis of beta beta' subunits of RNA polymerase has been examined through the use of a polyamine-requiring mutant of Escherichia coli. Results from autoradiographic estimation and activity measurement of RNA polymerase in cell extracts prepared from polyamine starved and unstarved bacteria showed that polyamines stimulate the synthesis of beta beta' subunits of RNA polymerase 2- to 3.6-fold.