Linked Life Data

10748161

Source:http://linkedlifedata.com/resource/pubmed/id/10748161

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
21
pubmed:dateCreated
2000-6-30
pubmed:abstractText
Cys(38) and Cys(280) of p66/p51 human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) can be converted to Ser without affecting enzyme function. We have exploited this feature to construct and purify "monocysteine" RT derivatives for site-specific modification with the photoactivable cross-linking agent, p-azidophenacyl bromide. Acylation of a unique cysteine residue introduced at the extreme C terminus of the p66 subunit (C(561)) with an azidophenacyl group allowed us to probe contacts between residues C-terminal to alpha-helix E' of the RNase H domain and structurally divergent nucleic acid duplexes. In a binary complex of RT and template-primer, we demonstrate efficient cross-linking to primer nucleotides -21 to -24/-25, and template nucleotides -18 to -21. Cross-linking specificity was confirmed by an analogous evaluation following limited primer extension, where the profile is displaced by the register of DNA synthesis. Finally, contact with a DNA primer hybridized to an isogenic RNA or DNA template indicates subtle alterations in cross-linking specificity, suggesting differences in nucleic acid geometry between duplex DNA and RNA/DNA hybrids at the RNase H domain. These data exemplify how site-specific acylation of HIV-1 RT can be used to provide high resolution structural data to complement crystallographic studies.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
May
pubmed:issn
0021-9258
pubmed:author
pubmed:issnType
Print
pubmed:day
26
pubmed:volume
275
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
16015-22
pubmed:dateRevised
2007-11-15
pubmed:meshHeading
pubmed:year
2000
pubmed:articleTitle
Probing contacts between the ribonuclease H domain of HIV-1 reverse transcriptase and nucleic acid by site-specific photocross-linking.
pubmed:affiliation
HIV Drug Resistance Program, Science Applications International Corporation, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Maryland 21072, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S.