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Pre-steady-state kinetic characterization of RNA-primed initiation of transcription by HIV-1 reverse transcriptase and analysis of the transition to a processive DNA-primed polymerization mode

Title data

Krebs, Ruth ; Wöhrl, Birgitta M. ; Cellai, Luciano ; Goody, Roger S. ; Restle, Tobias:
Pre-steady-state kinetic characterization of RNA-primed initiation of transcription by HIV-1 reverse transcriptase and analysis of the transition to a processive DNA-primed polymerization mode.
In: Biochemistry. Vol. 37 (1998) Issue 38 . - pp. 13349-13358.
ISSN 1520-4995
DOI: https://doi.org/10.1021/bi981102t

Abstract in another language

Single-turnover and equilibrium measurements were carried out to determine the basis of the apparently slow, nonprocessive polymerization reaction catalyzed by HIV-1 reverse transcriptase (RT) during transcription initiation, when both the primer and template are composed of RNA. Comparison of the binding and kinetic parameters of a 20-mer, all-RNA primer/35-mer template substrate to one identical in sequence but composed of a 20-mer, all-DNA primer/35-mer RNA template reveals striking differences. Equilibrium titrations yielded a dissociation constant (Kd) >200 nM for the RNA/RNA-RT complex which is at least 200-fold higher than that of the DNA/RNA-substrate (Kd approximately 1 nM). The affinity of the RT-RNA/RNA complex for dTTP was found to be at least 500 times lower (Kd approximately 3.4 mM) than that of the RT-DNA/RNA complex (Kd approximately 6.6 microM). The single-turnover dNTP incorporation time course using the RNA-primer substrate, the DNA-primer substrate, or a series of RNA-primer substrates preextended with one to eight deoxynucleotides showed that dNTP incorporation occurs with a biphasic exponential burst of +1 extension product, followed by a linear phase. At least three different RT-bound forms of the p/ts exist: a fast, kinetically competent form (single-turnover rate approximately 10-50 s⁻¹); a slow form (rate approximately 0.3-1 s⁻¹); and a form that is dead-end (no turnover). The studies further revealed that a switch to a fast, kinetically competent p/t occurs after six dNTPs are incorporated into the RNA primer, with the switch being defined as the transition from a minority to a majority of the p/t bound in the optimal manner.

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: 20 May 2019 06:31
Last Modified: 20 May 2019 06:31
URI: https://eref.uni-bayreuth.de/id/eprint/48993