Identification of the Primer Binding Domain in Human Immunodeficiency Virus Reverse Transcriptase

Amaresh Basu, Kiranjit K. Ahluwalia, Subhalakshmi Basu, Mukund J. Modak

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Abstract

We have labeled the primer binding domain of HIV1-RT with 5ʹ-32P-labeled (dT)15primer 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 tRNALys, inhibit p(dT)15binding 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 tRNALysinhibits the polymerase activity on poly(rA)↔(dT)15template-primer as well as the cross-linking of p(dT)15to the enzyme to a similar extent. At an enzyme to p(dT)15ratio 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)15was 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.

Original languageEnglish (US)
Pages (from-to)616-623
Number of pages8
JournalBiochemistry
Volume31
Issue number2
DOIs
StatePublished - Feb 1 1992

All Science Journal Classification (ASJC) codes

  • Biochemistry

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