RNA footprint mapping of RNA polymerase II molecules stalled in the intergenic region of polyomavirus DNA.
AUTOR(ES)
Brabant, F
RESUMO
RNA polymerase II molecules that transcribe the late strand of the 5.3-kb circular polyomavirus genome stall just upstream of the DNA replication origin, in a region containing multiple binding sites for polyomavirus large T antigen. Stalling of RNA polymerases depends on the presence of functional large T antigen and on the integrity of large T antigen binding site A. To gain insight into the interaction between DNA-bound large T antigen and RNA polymerase II, we mapped the position of stalled RNA polymerases by analyzing nascent RNA chains associated with these polymerases. Elongation of RNA in vitro, followed by hybridization with a nested set of DNA fragments extending progressively farther into the stalling region, allowed localization of the 3' end of the nascent RNA to a position 5 to 10 nucleotides upstream of binding site A. Ribonuclease treatment of nascent RNAs on viral transcription complexes, followed by in vitro elongation and hybridization, allowed localization of the distal end of stalled RNA polymerases to a position 40 nucleotides upstream of binding site A. This RNA footprint shows that elongating RNA polymerases stall at a site very close to the position of DNA-bound large T antigen and that they protect approximately 30 nucleotides of nascent RNA against ribonuclease digestion.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=189184Documentos Relacionados
- Mutation of large T-antigen-binding site A, but not site B or C, eliminates stalling by RNA polymerase II in the intergenic region of polyomavirus DNA.
- RNA polymerase II stalled at a thymine dimer: footprint and effect on excision repair.
- Electron microscopic mapping of wheat germ RNA polymerase II binding sites on cloned CaMV DNA.
- Transcriptional mapping of the ribosomal RNA region of mouse L-cell mitochondrial DNA.
- Characterization of tRNA genes in tRNA region II of yeast mitochondrial DNA.