Biochemistry. 1992 Jan 21;31(2):616-23. Unique Identifier : AIDSLINE
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.
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/CHEMISTRY Support, Non-U.S. Gov't Support, U.S.
Gov't, P.H.S. Templates JOURNAL ARTICLE