Identification of the primer binding domain in human immunodeficiency virus reverse transcriptase

Biochemistry. 1992 Jan 21;31(2):616-23. doi: 10.1021/bi00117a045.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Chromatography, DEAE-Cellulose
  • Chromatography, High Pressure Liquid
  • Cross-Linking Reagents
  • HIV-1 / enzymology*
  • Molecular Sequence Data
  • Oligodeoxyribonucleotides / chemistry*
  • Protein Binding
  • Protein Conformation
  • RNA-Directed DNA Polymerase / analysis*
  • RNA-Directed DNA Polymerase / chemistry
  • Templates, Genetic

Substances

  • Cross-Linking Reagents
  • Oligodeoxyribonucleotides
  • RNA-Directed DNA Polymerase