Eukaryotic ternary transcription complexes. II. An approach to the determination of chromatin conformation at the site of transcription.
Digestion of rat liver nuclei by endogenous nucleases or micrococcal nuclease releases a chromatin fraction containing RNA polymerases I and II bound to DNA fragments in ternary transcription complexes. To label the DNA in these transcription complexes, the polymerases were allowed to add radioactively labelled ribonucleotides in vitro to in vivo-initiated RNA chains. During this transcription step, nucleic acids were photochemically cross-linked using 8-methoxypsoralen. Nucleic acids in transcription complexes were then sized by gel electrophoresis. Under conditions where RNA polymerases I and II were active in vitro, most of the labelled DNA was found in a series of fragments of sizes which were multiples of approximately 200 base-pairs. When polymerase I alone was active, the smallest member of this series carried the bulk of the label; when polymerase II also was active, a significant proportion of the label was carried on the dimer and higher oligomers. Proteins other than polymerase alone are shown to be responsible for the pattern of DNA fragments protected from nucleases. Therefore active RNA polymerases I and II in vivo are in close proximity to structures protecting DNA fragments, the sizes of which are similar to those found in nucleosomes. We have yet to establish that these structures are composed of histones.
D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D002843:Chromatin / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D008099:Liver / Q000378:metabolism; D008730:Methoxsalen / Q000494:pharmacology; D008836:Micrococcal Nuclease / Q000378:metabolism; D012318:RNA Polymerase I / Q000378:metabolism*; D012319:RNA Polymerase II / Q000378:metabolism*; D051381:Rats; D014158:Transcription, Genetic
D002843:Chromatin; D012319:RNA Polymerase II; D012321:DNA-Directed RNA Polymerases; D012318:RNA Polymerase I; D008836:Micrococcal Nuclease; D008730:Methoxsalen