Photoaffinity labeling by 4-thiodideoxyuridine triphosphate of the HIV-1 reverse transcriptase active site during synthesis. Sequence of the unique labeled hexapeptide.
The active site of HIV-1 reverse transcriptase (HIV-1 RT) was investigated by photoaffinity labeling based on catalytic competence. A stable ternary elongation complex was assembled containing enzyme, DNA template (RT20), DNA primer molecule (P12), and the necessary dNTPs (one of which was alpha-32P-labeled) needed for primer elongation. The photoaffinity probe 4-thiodideoxyuridine triphosphate was incorporated uniquely at the 3' terminus of the 32P-labeled DNA product. Upon photolysis, the p66 subunit of a HIV-1 RT heterodimer (p66/p51) was uniquely cross-linked to the DNA product and subsequently digested by either trypsin or endoproteinase Lys-C. The labeled HIV-1 RT peptide was separated, purified, and finally subjected to Edman microsequencing. A unique radioactive hexapeptide (V276RQLCK281) was identified and sequenced. Our photoaffinity labeling results were positioned on the HIV-1 RT. DNA.Fab complex x-ray crystallography structure and compared with the suggested aspartic triad active site.
Lin
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Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, USA.
Henzel
W J
WJ
Nayak
S
S
Dennis
D
D
D001665:Binding Sites; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D054303:HIV Reverse Transcriptase / Q000737:chemistry / Q000378:metabolism*; D009842:Oligopeptides / Q000737:chemistry*; D010446:Peptide Fragments / Q000737:chemistry; D019920:Photoaffinity Labels; D013873:Thionucleotides / Q000737:chemistry*; D014544:Uridine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry
C110098:4-thiodideoxyuridine triphosphate; D009842:Oligopeptides; D010446:Peptide Fragments; D019920:Photoaffinity Labels; D013873:Thionucleotides; D054303:HIV Reverse Transcriptase; D014544:Uridine Triphosphate