Chromatin RNA polymerase activity from soybean hypocotyls treated with gibberellic acid and AMO-1618

Chromatin isolated from control, AMO-1618 [2′-isopropyl-4′-(trimethylammonium chloride)-5′-methylphenyl piperidine carboxylate] and gibberellic acid (GA) treated soybean hypocotyl tissue incorporates labeled nucleoside triphosphates into acid-insoluble RNA. Gibberellic acid, sprayed on intact soybean hypocotyls, is shown to have enhanced the level of chromatin RNA polymerase activity while chromatin isolated from hypocotyls pretreated with AMO-1618 exhibits a lower polymerase activity relative to the control. Chromatin extracted from the treated or untreated seedlings are all sensitive to the inhibition (in varying degrees) by the presence of actinomycin D, pyrophosphate, or ribonuclease. Thus enhanced (or decreased) RNA-synthesizing capacity of chromatin in response to chemical treatments may be due to enhanced (or decreased) synthesis of RNA polymerase.     

RNA polymerase II acts as an RNA‐dependent RNA polymerase to extend and destabilize a non‐coding RNA

RNA polymerase II (Pol II) is a well‐characterized DNA‐dependent RNA polymerase, which has also been reported to have RNA‐dependent RNA polymerase (RdRP) activity. Natural cellular RNA substrates of mammalian Pol II, however, have not been identified and the cellular function of the Pol II RdRP activity is unknown. We found that Pol II can use a non‐coding RNA, B2 RNA, as both a substrate and a template for its RdRP activity. Pol II extends B2 RNA by 18 nt on its 3′‐end in an internally templated reaction. The RNA product resulting from extension of B2 RNA by the Pol II RdRP can be removed from Pol II by a factor present in nuclear extracts. Treatment of cells with α‐amanitin or actinomycin D revealed that extension of B2 RNA by Pol II destabilizes the RNA. Our studies provide compelling evidence that mammalian Pol II acts as an RdRP to control the stability of a cellular RNA by extending its 3′‐end.     

Structure and function of lineage-specific sequence insertions in the bacterial RNA polymerase beta' subunit

The large beta and beta' subunits of the bacterial core RNA polymerase (RNAP) are highly conserved throughout evolution. Nevertheless, large sequence insertions in beta and beta' characterize specific evolutionary lineages of bacteria. The Thermus aquaticus RNAP beta' subunit contains a 283 residue insert between conserved regions A and B that is found in only four bacterial species. The Escherichia coli RNAP beta' subunit contains a 188 residue insert in the middle of conserved region G that is found in a wide range of bacterial species. Here, we present structural studies of these two beta' insertions. We show that the inserts comprise repeats of a previously characterized fold, the sandwich-barrel hybrid motif (as predicted from previous sequence analysis) and that the inserts serve significant roles in facilitating protein/protein and/or protein/nucleic acid interactions.     

Feedback regulation of RNA polymerase subunit synthesis after the conditional overproduction of RNA polymerase in Escherichia coli

Summary The and subunit of RNA polymerase are thought to be controlled by a translational feedback mechanism regulated by the concentration of RNA polymerase holoenzyme. To study this regulation in vivo, an inducible RNA polymerase overproduction system was developed. This system utilizes plasmids from two incompatibility groups that carry RNA polymerase subunit genes under lac promoter/operator control. When the structural genes encoding the components of core RNA polymerase (, and ) or holoenzyme (, , and ⁷⁰) are present on the plasmids, induction of the lac promoter results in a two fold increase in the concentration of functional RNA polymerase. The induction of RNA polymerase overproduction is characterized by an initial large burst of synthesis followed by a gradual decrease as the concentration of RNA polymerase increases. Overproduction of RNA polymerase in a strain carrying an electrophoretic mobility mutation in the rpoB gene results in the specific repression of synthesis off the chromosome. These results indicate that RNA polymerase feedback regulation controls synthesis in vivo.     

Real-time quantification of RNA polymerase activity using a "broken beacon"

A novel assay using a hybridization-based method was developed for real-time monitoring of RNA synthesis. In this work, a "broken beacon" in which the fluor and quencher were located on separate but complementary oligonucleotides was used to quantify the amount of RNA production by T7 polymerase. The relative lengths of the fluor-oligo and quencher-oligo, and their relative concentrations were optimized. The experimentally determined limit-of-detection was approximately 45 nM. The new assay was compared to the "gold-standard" radiolabel ([(32)P]NTP incorporation) assay for RNA quantification. While the broken beacon assay exhibited a higher limit of detection, it provided an accurate measure of RNA production rates. However, the broken beacon assay provided the significant analytical advantages of (i) a real-time and continuous measurement, (ii) no requirement for the use of radiolabels or gel-based analysis, and (iii) substantial time and labor savings.     

RNA template-mediated inhibition of hepatitis C virus RNA-dependent RNA polymerase activity

The enzymatic activity of hepatitis C virus (HCV) RNA-dependent RNA polymerase NS5B is modulated by the molar ratio of NS5B enzyme and RNA template. Depending on the ratio, either template or enzyme can inhibit activity. Inhibition of NS5B activity by RNA template exhibited characteristics of substrate inhibition, suggesting the template binds to a secondary site on the enzyme forming an inactive complex. Template inhibition was modulated by primer. Increasing concentrations of primer restored NS5B activity and decreased the affinity of template for the secondary site. Conversely, increasing template concentration reduced the affinity of primer binding. The kinetic profiles suggest template inhibition results from the binding of template to a site that interferes with primer binding and the formation of productive replication complexes.     

Chromatographic separation of DNA dependent RNA polymerases and molecular properties of RNA polymerase II from a Leishmania Spp

Multiple forms of DNA-dependent RNA polymerases have been isolated and characterized from Leishmania strain UR6 promastigotes. RNA polymerases from this organism fail to resolve into multiple forms by conventional chromatography on DEAE-Sephadex A25, but could be separated by a modification of the method using CM-Sephadex C25. The CM-Sephadex bound enzyme is resistant toamanitin even up to a concentration of 250g/ml. The activity which flows through CM-Sephadex further resolves into two forms upon chromatography on DEAE-Sephadex A25. These forms are sensitive to -amanitin to different extent. Enzyme activity in peak I is 50% inhibited by 3g/ml and in peak II by 50g/ml of the drug respectively. The enzyme in peak I has been further purified by heparin agarose and fast performance liquid chromatography (FPLC) on MonoQ. The enzyme has Stoke's radius of 70å, a sedimentation coefficient of 17.6S and an f/fo of 1.35. Analysis of ammonium sulfate and met n peak I, relative activities with Mn+2 versus Mg+2 and template specificities gave results similar to those reported for other type II RNA polymerases in eukaryotes. The MonoQ purified enzyme resolves into 16 polypeptides on denaturing polyacrylamide gel and densitometric analysis suggests that 9 major bands are present in the stoichiometry expected of RNA polymerase subunits having molecular weights: 154000; 104000; 77000; 64000; 52000; 48000; 46000; 45000 and 39000 respectively.     

RNA polymerase activity in germinating onion seeds

Frozen sections of endosperm cut from dry unimbibed onion seed were immersed in an aqueous solution of tritium labelled triphosphate; nucleolar RNA polymerase (ribonucleoside triphosphate: RNA nucleotidyltransferase E.C. 2.7.7.6) activity was detected by autoradiography after soaking for 10–15 min in the solution of the radioactive nucleotide. Throughout germination, activity appears to be mainly confined to the nucleolus with chromatin incorporation being very low or non-existent. In the embryo, in contrast to the endosperm, chromatin activity is initiated after 1 hr presoaking, while the nucleolus displays a lag of several hours. No incorporation could be detected in vivo before 18 hr.     

RNA polymerase activity in isolated Triticum aestivum embryos during germination

A gradual decrease in the total activity of DNA-dependent RNA polymerase in isolated wheat embryos began 6 hr after germination and continued for up to 48 hr. DEAE-cellulose column chromatography indicated the presence of two RNA polymerase fractions (major and minor) in the resting embryos, only one of which (major) could be detected in the embryos germinated for 48 hr. The major RNA polymerase fraction was tentatively identified as nucleoplasmic (RNA polymerase II).     

Targeted mutagenesis identifies Asp-569 as a catalytically critical residue in T7 RNA polymerase

In order to look more closely at a well-conserved region in T7 RNA polymerase (T7 RNAP) containing, as shown earlier, the functionally essential residues Pro-563 and Tyr-571, we used targeted mutagenesis to change those residues within this region that are invariant in all single-subunit RNA polymerases, and characterized the mutant enzymes in vitro. The most interesting finding of this study was the crucial importance of the acidic group of Asp-569. In addition, we have shown that the phenolic ring is the most significant functional group of Tyr-571, with the hydroxy group also contributing to promoter binding.     

Heat shock response in Escherichia coli promotes assembly of plasmid encoded RNA polymerase beta-subunit into RNA polymerase

Summary Escherichia coli cells, carrying a rifampicin sensitive RNA polymerase -subunit gene in the chromosome and a rifampicin resistant -subunit gene placed under the control of a strong promoter in a multicopy plasmid, are unable to grow in the presence of rifampicin, despite the accumulation of large quantities of the resistant subunit. A major portion of the overproduced subunit is found in an insoluble form. Conditions known to induce the heat shock proteins (hsps), e.g. elevated temperature or the presence of ethanol in the growth medium, increase the amount of the plasmid-borne -subunit which apparently assembles into active RNA polymerase and makes the plasmid bearing cells rifampicin resistant. Alternatively, plasmid-borne subunits assemble into RNA polymerase with low efficiency in rpoH mutant cells known to have reduced level of hsps. We suggest that the plasmid-borne subunit is poorly assembled into RNA polymerase and that hsps promote the assembly by interfering with -subunit aggregation.