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.
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.
Wöhrl
B M
BM
Abteilung Physikalische Biochemie, Max-Planck-Institut für Molekulare Physiologie, Otto-Hahn-Strasse 11, Dortmund, 44227, Germany. birgit.woehrl@mpi-dortmund.mpg.de
Krebs
R
R
Goody
R S
RS
Restle
T
T
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
D017931:DNA Primers; D004268:DNA-Binding Proteins; D009711:Nucleotides; D012313:RNA; D004247:DNA; D054303:HIV Reverse Transcriptase