Active site labeling of HIV-1 reverse transcriptase
- 1 May 1993
- journal article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 32 (18), 4938-4942
- https://doi.org/10.1021/bi00069a031
Abstract
The human immunodeficiency virus-1 reverse transcriptase (HIV-1 RT) heterodimer (M(r) = 66,000 and M(r) = 51,000) has been photoaffinity labeled using 4-thiodeoxyuridine triphosphate (S4-dUTP) as a probe. A nascent polymerization complex was assembled from a single-stranded DNA template, a 12-mer DNA primer, and the necessary dNTPs (one of which was alpha-32P-labeled) to extend the primer to produce the n-1 product. The photoaffinity probe was then uniquely added at the 3'-terminal position of the extended primer bound at the catalytic site and photolyzed. The larger subunit (p66) was exclusively derivatized. The unique radioactive peptide resulting from proteolysis was isolated and identified by amino acid sequencing.Keywords
This publication has 8 references indexed in Scilit:
- Wheat germ and yeast RNA polymerase II: Photoaffinity labeling by 4-thiouracil 5'-monophosphate positioned uniquely at the 3' end of an enzyme-bound phosphorus-32-containing transcriptBiochemistry, 1993
- Active site studies of human immunodeficiency virus reverse transcriptaseBiochemistry, 1992
- Localization of a polynucleotide binding region in the HIV-1 reverse transcriptase: implications for primer bindingBiochemistry, 1991
- Crosslinking of substrates occurs exclusively to the p66 subunit of heterodimeric HIV-1 reverse transcriptaseBiochemical and Biophysical Research Communications, 1991
- Substrate Binding in Human Immunodeficiency Virus Reverse TranscriptasePublished by Elsevier ,1989
- Human immunodeficiency virus 1 reverse transcriptasePublished by Elsevier ,1989
- A System for Rapid DNA Sequencing with Fluorescent Chain-Terminating DideoxynucleotidesScience, 1987
- End Labeling of Synthetic ProbesPublished by Elsevier ,1986