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.
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.
Thrall
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SH
Max-Planck-Institut für molekulare Physiologie, Abteilung Physikalische Biochemie, Rheinlanddamm 201, 44139 Dortmund, Germany.
Krebs
R
R
Wöhrl
B M
BM
Cellai
L
L
Goody
R S
RS
Restle
T
T
D001482:Base Composition; D001665:Binding Sites; D002384:Catalysis; D017931:DNA Primers / Q000737:chemistry*; D004279:DNA, Viral / Q000138:chemical synthesis / Q000737:chemistry*; D003854:Deoxyribonucleotides / Q000737:chemistry; D054303:HIV Reverse Transcriptase / Q000737:chemistry*; D007700:Kinetics; D012313:RNA / Q000138:chemical synthesis; D012367:RNA, Viral / Q000138:chemical synthesis; D013379:Substrate Specificity; D013698:Templates, Genetic; D014158:Transcription, Genetic
D017931:DNA Primers; D004279:DNA, Viral; D003854:Deoxyribonucleotides; C034477:RNA primers; D012367:RNA, Viral; D012313:RNA; D054303:HIV Reverse Transcriptase