Multiple DNA-dependent RNA polymerases in Trypanosoma cruzi.

DNA-dependant RNA polymerase solubilized from cultured Trypanosoma cruzi epimastigotes was chromatographed on A-25 Sephadex and gave a single peak of activity. Subsequent phosphocellulose chromatography revealed two peaks of RNA polymerase activity. These peaks have different sensitivities to the toxin alpha-amanitin. The first peak is 50% inhibited by 17.8 micrograms ml-1 amanitin while the second peak is 50% inhibited by an amanitin concentration of 44.6 micrograms ml-1. The activity of both peaks is blocked by actinomycin D, but is unaffected by rifampicin. Each peak is stimulated by Mn2+, and is optimal with single stranded DNA as a template.

Use of RNA polymerase as an enzymatic probe of nucleosomal structure.

Nucleosomes prepared from human placental nuclei and Escherichia coli DNA-dependent RNA polymerase (nucleoside triphosphate: RNA nucleotidyl transferase EC.2.7.7.6) form stable initiation complexes. This property is utilized as a probe of nucleosome structure. RNA polymerase initiation has been studied on purified nucleosomes, nucleosome cores, and nucleosomal DNA. The affinity of E. coli RNA polymerase for both nucleosome cores and monomers was 5-6 fold less than found for nucleosomal DNA. No difference in apparent initiation Km was found between cores and mononucleosomes. This suggests that initiation does not preferentially occur on the DNA tails of nucleosomes. Once initiated and allowed to form nascent RNA, these complexes are very stable to ionic strength changes. Under conditions in which free enzyme is inactivated with rifampicin, the enzyme in the complex retains activity as demonstrated by its ability to transcribe and reinitiate on both nucleosomes and free DNA. These complexes can be well resolved from free nucleosomes on preparative polyacrylamide gels and both can be eluted from gels for analysis of proteins and DNA sequence complexity. Studies using (125I) labelled nucleosomes show that histones are retained in the initiation complex, and are not dissociated by the enzyme during initiation.

Transcription of nucleosomes from human chromatin.

Nucleosomes (chromatin subunits) prepared by micrococcal nuclease digestion of human nuclei are similar in histone content but substantially reduced in non-histone proteins as compared to undigested chromatin. Chromatin transcription experiments indicate that the DNA in the nucleosomes is accessible to DNA-dependent RNA polymerase in vitro. The template capacities of chromatin and nucleosomes are 1.5 and 10%, respectively, relative to high molecular weight DNA, with intermediate values for oligonucleosomes. Three distinct sizes of transcripts, 150, 120 and 95 nucleotides in length, are obtained when nucleosomes are used as templates. However, when nucleosomal DNA is used as a template, the predominant size of transcripts is 150 nucleotides. When oligonucleosomes are used as templates longer transcripts are obtained. This indicates that RNA polymerase can transcribe the DNA contained in the nucleosomes.

Purification using polyethylenimine precipitation and low molecular weight subunit analyses of calf thymus and wheat germ DNA-dependent RNA polymerase II.

DNA-dependent RNA polymerase II from calf thymus has been successfully purified using polythylenimine precipitation. Thus, 5-6 mg of nearly homogeneous homogeneous trna polymerase II (greater than 96% pure) can be prepared from 1 kg of calf thymus with three chromatography steps following extraction and precipitation of the enzyme from the polyethylenimine pellet. This procedure eliminates the high salt extraction of chromatin previously used in purification of this enzyme and makes possible the large scale preparation of mammalian RNA polymerase II. Calf thymus polymerase II prepared by this method is greater than 90% form IIb and consists of ten different subunits having the following molecular weights: 180 000; 145 000; 36 000; 25 000; 20 000; 18 500; 16 000; 15 000; 12 000; 11 500. The homologous enzyme isolated from wheat germ is greater than 90% form IIa and contains subunits of the following molecular weights: 206 000; 145 000; 44 000-47 000; 24 500; 21 000; 19 000; 17 000; 14 000; 13 500. The wheat germ and calf thymus enzymes exhibit similar subunits structures, but the molecular weights of individual subunits are clearly different between the enzymes. Wheat germ RNA polymerase II is 50% inhibited by 0.271 microng/mL of alpha-amanitin, a level 30-fold higher than that found for calf thymus RNA polymerase II. These enzymes are further distinguished by the absence of antigenic cross reactivity.