Linked Life Data

2464595

Source:http://linkedlifedata.com/resource/pubmed/id/2464595

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
6
pubmed:dateCreated
1989-3-21
pubmed:abstractText
DNA fragments containing the adenovirus 2 major late or simian virus 40 early promoters were attached to a solid support via a biotin-streptavidin linkage at one end of the fragment, upstream of the RNA start site. Templates immobilized in this manner were incubated with HeLa cell nuclear extracts to form preinitiation complexes containing RNA polymerase II and accessory proteins required for faithful in vitro transcription. These complexes did not require ATP or dATP for assembly, were sensitive to 0.25% Sarkosyl, and were stable to extensive washing. Their incubation with specific combinations of nucleoside triphosphates resulted in the initiation of RNA chain polymerization in situ, while addition of the remaining nucleoside triphosphates was necessary to produce a full length runoff RNA. Transcriptional activity associated with preinitiation complexes was purified approximately 300-fold, relative to the unfractionated nuclear extract. The use of immobilized template permits considerable flexibility in experimental design, as substrates and inhibitors can be added and washed out of the reaction at each step. We exploited this property of the system to dissect the temporal substrate requirements for initiation of RNA synthesis. It is known from prior work that at least one step in the promoter-dependent RNA synthesis reaction requires an adenosine nucleotide that is hydrolyzable at the beta, gamma-position. This requirement is independent of the initiating nucleotide and can be satisfied by dATP, which is not ordinarily incorporated into the RNA product. We show here that the beta, gamma-hydrolyzable adenosine nucleotide must be present simultaneously with the initiating nucleoside triphosphates. No reaction occurred when complexes were incubated with dATP, washed to remove dATP, and incubated subsequently with the two initiating nucleotides.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Feb
pubmed:issn
0021-9258
pubmed:author
pubmed:issnType
Print
pubmed:day
25
pubmed:volume
264
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
3223-9
pubmed:dateRevised
2008-11-21
pubmed:meshHeading
pubmed-meshheading:2464595-Adenine Nucleotides, pubmed-meshheading:2464595-Adenosine Triphosphate, pubmed-meshheading:2464595-Adenoviridae, pubmed-meshheading:2464595-Cell Nucleus, pubmed-meshheading:2464595-DNA, Viral, pubmed-meshheading:2464595-Deoxyadenine Nucleotides, pubmed-meshheading:2464595-Detergents, pubmed-meshheading:2464595-Electrophoresis, Polyacrylamide Gel, pubmed-meshheading:2464595-Enzymes, Immobilized, pubmed-meshheading:2464595-HeLa Cells, pubmed-meshheading:2464595-Humans, pubmed-meshheading:2464595-Nucleotides, pubmed-meshheading:2464595-Promoter Regions, Genetic, pubmed-meshheading:2464595-RNA, pubmed-meshheading:2464595-RNA Polymerase II, pubmed-meshheading:2464595-Simian virus 40, pubmed-meshheading:2464595-Templates, Genetic, pubmed-meshheading:2464595-Transcription, Genetic
pubmed:year
1989
pubmed:articleTitle
Promoter-dependent transcription by RNA polymerase II using immobilized enzyme complexes.
pubmed:affiliation
Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder 80309.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, Non-U.S. Gov't