Identification of the primer binding domain in human immunodeficiency virus reverse transcriptase.
We have labeled the primer binding domain of HIV1-RT with 5'-32P-labeled (dT)15 primer using ultraviolet light energy. The specificity of the primer cross-linking to HIV1-RT was demonstrated by competition experiments. Both synthetic and natural primers, e.g., p(dA)15, p(dC)15, and tRNA(Lys), inhibit p(dT)15 binding and cross-linking to the enzyme. The observed binding and cross-linking of the primer to the enzyme were further shown to be functionally significant by the observation that tRNA(Lys) inhibits the polymerase activity on poly(rA).(dT)15 template-primer as well as the cross-linking of p(dT)15 to the enzyme to a similar extent. At an enzyme to p(dT)15 ratio of 1:3, about 15% of the enzyme can be cross-linked to the primer. To identify the domain cross-linked to (dT)15, tryptic peptides were generated and purified by a combination of HPLC on a C-18 reverse-phase column and DEAE-Sephadex chromatography. A single peptide cross-linked to p(dT)15 was identified. This peptide corresponded to amino acid residues 288-307 in the primary sequence of HIV1-RT as judged by amino acid composition and sequence analyses. Further, Leu(289)-Thr(290) and Leu(295)-Thr(296) of HIV1-RT appear to be the probable sites of cross-linking to the primer p(dT)15.
Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey, Newark 07103-2714.
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Basu
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Ahluwalia
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Basu
M J
MJ
Modak
D000595:Amino Acid Sequence; D002848:Chromatography, DEAE-Cellulose; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D015497:HIV-1 / Q000201:enzymology*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D012194:RNA-Directed DNA Polymerase / Q000032:analysis* / Q000737:chemistry; D013698:Templates, Genetic
D003432:Cross-Linking Reagents; D009838:Oligodeoxyribonucleotides; D012194:RNA-Directed DNA Polymerase