title: Refined model for primer/template binding by HIV-1 reverse transcriptase: pre-steady-state kinetic analyses of primer/template binding and nucleotide incorporation events distinguish between different binding modes depending on the nature of the nucleic acid substrate.
abstract: The kinetic mechanism of nucleic acid substrate binding and nucleotide incorporation by human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) was analysed using synthetic DNA/DNA and DNA/RNA primer/templates (p/t) without predicted secondary structures in the single-stranded region. Determination of the pre-steady-state kinetics of p/t binding by a combination of stopped-flow and quench flow methods indicate a branched binding mechanism for the HIV-1 RT/nucleic acid interaction. Analysis of p/t-RT association by stopped-flow measurements suggest a three-step binding mode with an initial second-order step followed by two isomerisation steps with rates of about 6 s(-1)and 0.5 s(-1), respectively. Determination of the rate-limiting step of the association process via single turnover, single nucleotide incorporation analysis by quench flow measurements revealed two binding events (the initial second-order step cannot be detected with this experimental set-up) with rates of 4 - 7 s(-1)and 0.4 - 0. 7 s(-1), respectively, indicating that both binding events exist in parallel. Thorough pre-steady-state analysis of single turnover, single nucleotide incorporation kinetics showed that dNTP incorporation occurs with a biphasic exponential burst followed by a linear phase. The exponential burst consists of a fast phase with rates of 20 - 60 s(-1) and a slow phase with rates of 0.5 - 2 s(-1), respectively. The relative distribution of these two burst amplitudes differs significantly depending upon which substrate is used. The DNA/RNA-RT complex shows primarily fast incorporation (>80 %) whereas less than 45 % of the DNA/DNA-RT complex incorporate dNTP rapidly. The same relative distribution of amplitudes concerning the two substrates is also found for the association process of RT and p/t. Analysis of dNTP incorporation of the preformed RT-p/t complex in the presence of a nucleic acid competitor shows no effect on the biphasic burst amplitude, however the linear phase disappears. Here, a refined model of the mechanism of RT-p/t binding is presented which is based on the suggestion that two different RT-p/t complexes are formed, i.e. a productive enzyme/substrate complex which is capable of nucleotide incorporation and a non-productive complex which has to undergo an isomerisation before dNTP incorporation can occur. In addition, binding of RT to its substrate can lead to a dead end complex that is not capable of dNTP incorporation.
authors.affiliation: Abteilung Physikalische Biochemie, Max-Planck-Institut für Molekulare Physiologie, Otto-Hahn-Strasse 11, Dortmund, 44227, Germany. birgit.woehrl@mpi-dortmund.mpg.de
authors.forename: B M
authors.initials: BM
authors.lastname: Wöhrl
authors.forename: R
authors.initials: R
authors.lastname: Krebs
authors.forename: R S
authors.initials: RS
authors.lastname: Goody
authors.forename: T
authors.initials: T
authors.lastname: Restle
mesh_terms: D001667:Binding, Competitive; D004247:DNA / Q000096:biosynthesis / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry*; D054303:HIV Reverse Transcriptase / Q000737:chemistry*; D006801:Humans; D009711:Nucleotides / Q000737:chemistry; D012313:RNA / Q000378:metabolism; D013698:Templates, Genetic
chemical_list: D017931:DNA Primers; D004268:DNA-Binding Proteins; D009711:Nucleotides; D012313:RNA; D004247:DNA; D054303:HIV Reverse Transcriptase