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.
D002467:Cell Nucleus / Q000032:analysis; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D005260:Female; D006801:Humans; D007700:Kinetics; D009707:Nucleosomes / Q000032:analysis*; D010920:Placenta / Q000032:analysis; D011247:Pregnancy; D014176:Protein Biosynthesis; D014158:Transcription, Genetic
D009707:Nucleosomes; D012321:DNA-Directed RNA Polymerases