Photoaffinity labeling by 4-thiodideoxyuridine triphosphate of the HIV-1 reverse transcriptase active site during synthesis. Sequence of the unique labeled hexapeptide.

The Journal of Biological Chemistry
S LinD Dennis

Abstract

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.

References

Apr 1, 1977·Nucleic Acids Research·C G Sahasrabuddhe, G F Saunders
Aug 25, 1992·Biochemistry·L L Mitchell, B S Cooperman
May 1, 1990·Protein Engineering·M DelarueP Argos
Jul 1, 1989·Proceedings of the National Academy of Sciences of the United States of America·B A LarderD J Purifoy
Aug 28, 1987·Journal of Chromatography·W J HenzelC Watanabe
Jan 1, 1994·Annual Review of Biochemistry·C M Joyce, T A Steitz
May 11, 1993·Biochemistry·N Sheng, D Dennis
Jul 1, 1993·Proceedings of the National Academy of Sciences of the United States of America·A Jacobo-MolinaP Clark
Jan 1, 1993·Annual Review of Biochemistry·K A Johnson
Feb 1, 1996·Biological Chemistry Hoppe-Seyler·M Hottiger, U Hübscher

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