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-I reverse transcriptase (HIV-1 RT) was investig ated by photoaffinity labeling based on catalytic competence, A stable ternary elongation complex was assembled containing enzyme, DNA templ ate (RT20), DNA primer molecule (P12), and the necessary dNTPs (one of which was alpha-P-32-labeled) needed for primer elongation. The photo affinity probe 4-thiodide-oxyuridine triphosphate was incorporated uni quely at the 3' terminus of the P-32-labeled DNA product, Upon photoly sis, the p66 subunit of a HIV-1 RT heterodimer (p66/p51) was uniquely cross-linked to the DNA product and subsequently digested by either tr ypsin or endoproteinase Lys-C. The labeled HIV-1 RT peptide was separa ted, purified, and finally subjected to Edman microsequencing. A uniqu e radioactive hexapeptide (V(276)RQLCK(281)) was identified and sequen ced, Our photoaffinity labeling results were positioned on the HIV-1 R T DNA Fab complex x-ray crystallography structure and compared with th e suggested aspartic triad active site.