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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

Title data

Wöhrl, Birgitta ; Krebs, Ruth ; Goody, Roger S. ; Restle, Tobias:
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.
In: Journal of Molecular Biology. Vol. 292 (1999) Issue 2 . - pp. 333-344.
ISSN 0022-2836
DOI: https://doi.org/10.1006/jmbi.1999.3057

Abstract in another language

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.

Further data

Item Type: Article in a journal
Refereed: Yes
Institutions of the University: Faculties > Faculty of Biology, Chemistry and Earth Sciences
Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry
Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry > Chair Biopolymers
Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry > Chair Biopolymers > Lehrstuhl Biopolymere - Apl. Prof. Dr. Birgitta Wöhrl
Result of work at the UBT: No
DDC Subjects: 500 Science > 540 Chemistry
500 Science > 570 Life sciences, biology
Date Deposited: 17 May 2019 07:41
Last Modified: 17 May 2019 07:41
URI: https://eref.uni-bayreuth.de/id/eprint/48977