Active site labeling of HIV-1 reverse transcriptase.
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.
Sheng
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Department of Chemistry and Biochemistry, University of Delaware, Newark 19716.
Dennis
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D000163:Acquired Immunodeficiency Syndrome / Q000201:enzymology*; D000345:Affinity Labels / Q000528:radiation effects; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000096:biosynthesis; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology*; D008969:Molecular Sequence Data; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism* / Q000528:radiation effects; D017421:Sequence Analysis; D014544:Uridine Triphosphate / Q000031:analogs & derivatives / Q000528:radiation effects
D000345:Affinity Labels; C059149:4-thiouridine triphosphate; D004247:DNA; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D014544:Uridine Triphosphate