Mapping of RNA polymerase on mammalian genes in cells and nuclei

Détails

ID Serval
serval:BIB_CBD09F7B3C09
Type
Article: article d'un périodique ou d'un magazine.
Collection
Publications
Titre
Mapping of RNA polymerase on mammalian genes in cells and nuclei
Périodique
Molecular Biology of the Cell
Auteur(s)
Mirkovitch  J., Darnell, J. E., Jr. 
ISSN
1059-1524 (Print)
Statut éditorial
Publié
Date de publication
10/1992
Volume
3
Numéro
10
Pages
1085-94
Notes
Journal Article
Research Support, U.S. Gov't, P.H.S. --- Old month value: Oct
Résumé
The assembly of an RNA polymerase II initiation complex at a promoter is associated with the melting of the DNA template to allow the polymerase to read the DNA sequence and synthesize the corresponding RNA. Using the specific single-stranded modifying reagent KMnO4 and a new genomic sequencing technique, we have explored the melted regions of specific genes in genomic DNA of whole cells or of isolated nuclei. We have demonstrated for the first time in vivo the melting in the promoter proximal transcribed region that is associated with the presence of RNA polymerase II complexes. An interferon-inducible gene, ISG-54, exhibited KMnO4 sensitivity over approximately 300 nucleotides downstream of the RNA initiation site in interferon-treated cells when the gene was actively transcribed but not in untreated cells where the gene was not transcribed. The extent of KMnO4 modification was proportional to transcription levels. The KMnO4 sensitivity was retained when nuclei were isolated from induced cells but was lost if the engaged polymerases were further allowed to elongate the nascent RNA chains ("run-on"). The sensitivity to KMnO4 in isolated nuclei was retained if the run-on incubation was performed in the presence of alpha-amanitin, which blocks progress of engaged polymerases. A similar analysis identified an open sequence of only approximately 30 bases just downstream of the start site of the transthyretin (TTR) gene in nuclei isolated from mouse liver, a tissue where TTR is actively transcribed. This abrupt boundary of KMnO4 sensitivity, which was removed completely by allowing engaged polymerases to elongate RNA chains, suggests that most polymerases transcribing this gene paused at about position +20. The possibility of mapping at the nucleotide level the position of actively transcribing RNA polymerases in whole cells or isolated nuclei opens new prospects in the study of transcription initiation and elongation.
Mots-clé
Animals Cell Nucleus/enzymology Chromosome Mapping DNA, Single-Stranded/genetics Hela Cells Humans Indicators and Reagents Liver/metabolism Mice Nucleic Acid Conformation Potassium Permanganate Prealbumin/genetics RNA/chemistry/genetics RNA Polymerase II/*genetics/metabolism Transcription, Genetic
Pubmed
Web of science
Création de la notice
25/01/2008 9:41
Dernière modification de la notice
03/03/2018 21:27
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