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Human immunodeficiency virus type 1 reverse transcriptase Affinity labeling of the primer binding site
Author(s) -
Mitina R.L.,
Doronin S.V.,
Dobrikov M.I.,
Tabatadze D.R.,
Levina A.S.,
Lavrik O.I.
Publication year - 1992
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(92)80945-d
Subject(s) - primer (cosmetics) , oligonucleotide , reverse transcriptase , covalent bond , chemistry , affinity labeling , enzyme , primer binding site , binding site , stereochemistry , microbiology and biotechnology , biochemistry , biology , rna , dna , organic chemistry , gene
Affinity 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 ArN 3 ‐d(U*T 9 ); the dependence of the rate of inactivation on primer concentration was found to be consistent with the K m 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 tRNA Ly5 .