Human immunodeficiency virus type 1 reverse transcriptase Affinity labeling of the primer binding site

AbstractAffinity modification of the primer site of HIV1-RT was performed with an oligonucleotide derivative containing a photoreactive azido group at the 5′ end of d(pT)10. The affinity of HIV1-RT for d(pT)10 and for its derivative was first estimated by measuring the Michaelis constants of these two oligonucleotides acting as primers in the retrotranscription of poly(rA). The enzyme was then inactivated under UV-irradiation at 303–365 nm in the presence of ArN3-d(U*T9); the dependence of the rate of inactivation on primer concentration was found to be consistent with the Km value. Last, selectivity of affinity modification was demonstrated through elongation of the covalently bound primer and selective protection of inactivation by d(pT)10 or tRNALy5