Active site labeling of HIV-1 reverse transcriptase.

N Sheng; D Dennis

doi:10.1021/bi00069a031

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Active site labeling of HIV-1 reverse transcriptase.

N Sheng·D Dennis

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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.

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Acquired Immunodeficiency Syndrome Affinity Labels Amino Acid Sequence Base Sequence Binding Sites DNA HIV Reverse Transcriptase HIV-1 Molecular Sequence Data RNA-Directed DNA Polymerase Sequence Analysis Uridine Triphosphate Journal Article

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N Sheng, D Dennis. 1993-05-11. Active site labeling of HIV-1 reverse transcriptase.. https://doi.org/10.1021/bi00069a031

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