Kinetic analysis of template.primer interactions with recombinant forms of HIV-1 reverse transcriptase

Biochemistry. 1993 Sep 21;32(37):9745-53. doi: 10.1021/bi00088a029.


The reverse transcriptase (RT) from the human immunodeficiency virus (HIV) exists predominantly as a heterodimer (p66/p51), but can also form a homodimer of p66 subunits (p66/p66). RT binds to template-primer (T/P) tightly to form the first complex in the reaction sequence poised to conduct DNA synthesis upon the addition of dNTP and Mg2+. We have made use of this property to kinetically analyze poly(rA)-(dT)n interactions with recombinant homo- and heterodimeric HIV-1 RT derived from HXB2R proviral DNA. A T/P challenge assay was used to quantitatively follow RT-T/P complex formation. The homo- and heterodimeric forms of RT bound to poly(rA)-(dT)16 in a kinetically similar fashion. There was no more than a 2-fold difference in kcat or for any T/P parameter examined: Km, Kd, kon, koff determined from a binary complex or from a complex incorporating dTMP, processivity, and stoichiometry of binding. In contrast, it was found that the T/P Km with heterodimeric RT derived from the NY5 strain was significantly greater than that determined for HXB2R enzyme, indicating that a kinetic diversity exists between RT derived from different viral strains. Since HXB2R RT binds to poly(rA)-(dT)16 tightly, Kd < 1 nM, active-site titrations are facilitated. At saturation, one T/P binds per two polypeptides, suggesting that RT binds substrate productively as a dimer and that if monomers are present they must rapidly form dimers in the presence of T/P.(ABSTRACT TRUNCATED AT 250 WORDS)

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Binding Sites
  • HIV Reverse Transcriptase
  • HIV-1 / classification
  • HIV-1 / enzymology*
  • Kinetics
  • Poly A / metabolism
  • Poly T / metabolism
  • RNA-Directed DNA Polymerase / metabolism*
  • Recombinant Proteins
  • Species Specificity
  • Structure-Activity Relationship
  • Templates, Genetic


  • Recombinant Proteins
  • Poly A
  • Poly T
  • HIV Reverse Transcriptase
  • RNA-Directed DNA Polymerase