Two species of RNA polymerase II released from rat liver chromatin (original) (raw)
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Journal of Molecular Biology, 1982
Cbromatin fragments of the RNA potymerase II-trans~iption~ complex were purified from the micrococcal m&ease digest of rat liver nuclei in the presence of n-butyrate, a potent histone deacetylase inhibitor. Polyacrylamide gel eiectrophoretic analysis in Triton acid-urea revealed that the extent of histone acetylation of the complex did not differ markedly from that of the total chromatin.
Biochimica et biophysica acta, 1981
Two forms of RNA polymerase II were released from rat liver chromatin by micrococcal nuclease digestion of the nuclei. One from behaved like a free RNA polymerase II and the other like a complex with other nuclear components. Both forms of RNA polymerase II activity were recovered in the 0.16 M NaCl-soluble fraction of the nuclear digest, and the complexed from the RNA polymerase II could transcribe its endogenous template under conditions permitting only of elongation of the RNA synthesis. The RNA polymerase II complex was further purified by gel filtration chromatography and column electrophoresis. Analysis of protein and DNA of the partially purified complex suggested that the RNA polymerase II was bound to mono- or dinucleosomes carrying some characteristic nonhistone proteins. Furthermore, in experiments on tissues from starved rats, the two forms of RNA polymerase II were found to originate from different functional states of the chromatin-bound enzyme in vivo.
A RNA-dependent RNA polymerase activity: implications for chromatin transcription experiments
Nucleic Acids Research, 1977
Mercurated nucleoside triphosphates have been used for transcription of chicken oviduct chromatin with E.coli RNA polymerase. The newly synthesized RNA was purified from preexisting RNA by SH-agarose chromatography and analyzed for the content of specific mRNA sequences. The apparent preferential production of ovalbumin mRNA sequences was not inhibited by actinomycin D, although total RNA synthesis was reduced by more than 90%. Furthermore, when globin mRNA alone, or added to oviduct chromatin, was incubated in the transcription assay, a significant fraction of this mRNA was retained on SH-agarose. The copurification of chromatin associated RNA with in vitro synthesized mercurated RNA was mainly due to a RNA-dependent synthesis of complementary sequences by the bacterial enzyme. Although denaturation of the transcripts prior to SH-agarose chromatography leads to a reduced contamination with endogenous ovalbumin specific RNA, we are unable to show that the messenger-specific RNA sequences purified with the newly mercurated RNA result from a DNA-dependent reaction.
Journal of Molecular Biology, 1978
A mild enzymic procedure for fractionating nuclei was based on a combination of light-scattering properties and electron microscopy in order to monitor the structural integrity of rat liver nuclei and to establish the gentlest conditions possible for their disruption. Incubation of nuclei with as little as 0.1 unit of micrococcal nuclease per ml for 60 seconds at 20 to 29"C, followed by EGTA, caused their total disruption with minimal perturbation of chromatin or transcriptional characteristics. A simple two-step differential centrifugation resolved the gently disrupted nuclei into three mechanically unsheared fractions. One of these (fraction P2) consisted of aggregates of 6 to 30 covalently linked nucleosomes, each containing about 200 base-pairs of DNA, and which are here termed polynucleoeomes. This fraction represented about 10% of nuclear DNA (200 to 6000 base-pairs), whose properties corresponded to euohromatin prepared by other methods. Since there is virtually no reinitiation of RNA synthesis in vitro by isolated nuclei or subnuclear preparations, endogenous RNA polymerase activities represent the elongation of RNA chains that were initiated in vivo. When the distribution of free (inactive in the absence of exogenous template) and endogenous template-engaged RNA polymerases I(A) and II(B) was monitored, the latter as an index of transcriptional complexes that existed in the intact nucleus, mild nuclease digestion was found not to alter the autonomous transcriptional characteristics of the disrupted nuclei. Over 85% of the template-engaged RNA polymerase II was recovered with polynucleosomes. Further nuclease digestion of this fraction showed that a minimum of six nucleosomesjunit was necessary for retaining the enzyme on its template. Polynucleosomes can therefore be considered as the basic structural units of chromatin which are selectively released by extremely mild enzymic treatment of whole nuclei from the transcriptionally active compartment and are capable of continuing *in vitro the elongation of RNA chains initiated in. viva.
Transcription of Rat-Liver Chromatin with Homologous Enzyme
Proceedings of the National Academy of Sciences, 1973
The product of transcription of rat-liver chromatin with homologous rat-liver Form-B polymerase in vitro is high molecular weight RNA with sedimentation coefficients principally in the range of 18-45 S. The average size is somewhat smaller than that of the heterogeneous high molecular weight RNA synthesized by the endogenous enzyme in vivo (or in isolated nuclei). We have excluded the possibility that this difference can be attributed to degradation of the nascent RNA occurring under the conditions of our experiments. The size of the RNA produced by the homologous enzyme nevertheless approaches that of the natural transcripts much more closely than does that of the RNA produced by bacterial RNA polymerases, which we, in addition to other authors, have found to sediment around 10 S.
Transcription by Eukaryotic RNA Polymerases A and B of Chromatin Assembled in vitro
European Journal of Biochemistry, 1979
Chromatin was assembled in vitro from simian virus 40 DNA form I and the calf-thymus four histones H2A, H2B, H3 and H4. Transcription with calf thymus RNA polymerases A and B (I and II) was greatly inhibited. Nucleosomes were found to inhibit both RNA chain initiation and elongation. The inhibition of elongation could be relieved by increasing ionic strength, suggesting that electrostatic interactions between histone octamer and DNA have to be broken for RNA polymerase to transcribe DNA organised into nucleosomes.
Nucleic Acids Research, 1984
Mononucleosomes obtained from cultured mouse hepatoma cells were incubated with RNA polymerase II from wheat germ. No free DNA was liberated as available templates under the experimental condition employed. Size analysis of the transcripts showed that the polymerase initiated transcription from either terminus and read through the DNA template of mononucleosomes. Sucrose density gradient centrifugation of the reaction mixture resolved mononucleosome-polymerase complexes from free materials. The complexes were characterized by the enrichment of DNA fragments containing the nucleosome linker region, the presence of H1 histone, and the increased susceptibility to DNase I. Both the complexes formed in the presence and absence of precursor nucleotides were susceptible. These suggest that RNA polymerase II prefers to bind to the linker region, and the polymerase-bound nucleosomes are structurally altered. The data were discussed in context with possible mechanisms of transcription of the nucleosome structure.
Sub-nuclear fractionation II. Intranuclear compartmentation of transcription in vivo and in vitro
Experimental Cell Research, 1974
DNA-dependent RNA polymerase activities were measured in subnuclear fractions obtained from rat liver by the procedure described in the preceding paper . Most of the total nuclear enzyme was recovered in a form bound to chromatin with only small amounts as free enzyme in the nucleoplasm. The multiple eukaryotic RNA polymerases were resolved according to the endogenous template to which they were bound and which they continue to transcribe in vitro. The A and B forms of the enzyme were distinguished from each other by their differential sensitivities to a-amanitin, exogenous native and denatured DNA, thermal denaturation at 45, Mg2+ and Mn2 ions, high ionic strength and by the binding of '*C-methyl-y-amanitin. RNA polymerase B (a-amanitin-sensitive) was exclusively recovered in the nucleoplasmic and euchromatin fractions. RNA polymerase A was recovered in the dispersed nucleolar as well as in heterochromatin. By assaying in the presence of cc-amanitin subnuclear fractions that had been pre-incubated at 45°C a third enzyme (form C) was located exclusively in heterochromatin fractions. Only the euchromatin associated RNA polymerase B was capable of initiating the synthesis of new RNA chains in vitro on endogenous template at low ionic strength. Raising the ionic strength abolished initiation but accelerated chain elongation by this form of enzyme.