HIV-1 reverse transcriptase interaction with model RNA-DNA duplexes.
HIV-1 reverse transcriptase (HIV-1 RT) is a multifunctional enzyme responsible for converting viral RNA into preintegrative DNA during the early stages of viral infection. DNA polymerase and RNase H activities are required, and several conformationally distinct primer-templates must be accommodated by the enzyme during the process. Parameters of interaction between model substrates (ligands) and HIV-1 RT (wild type p66/p51 and the RNase H-deficient mutant p66(E478Q)/p51) (analytes) were estimated by surface plasmon resonance at 25 degrees C, pH 8.0. Binding of RT to the ligands is specific and can be analyzed using a conventional 1:1 binding algorithm. RNA-DNA hybrids with 5'-template overhangs of 6 and 12 nucleotides bind to RT approximately one order of magnitude stronger than the corresponding 36-mer with blunt ends due to slower dissociation. Immobilization of the latter through either the 5'-end of RNA or DNA strand does not change the equilibrium constant (K(D)) for wild-type RT but the values of kinetic constants of association and dissociation differ significantly. For the p66(E478Q)/p51 enzyme, orientation effects are notable even altering the K(D) value. Binding of the p66(E478Q)/p51 to any RNA-DNA hybrids is slightly stronger compared with wild type. Data can be interpreted in terms of the mechanism of reverse transcription.
Gorshkova
I I
II
Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, NIH, Bethesda, Maryland 20892, USA.
Rausch
J W
JW
Le Grice
S F
SF
Crouch
R J
RJ
D000465:Algorithms; D001483:Base Sequence; D001667:Binding, Competitive; D019921:Biotinylation; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics / Q000378:metabolism; D054303:HIV Reverse Transcriptase / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology*; D007700:Kinetics; D008024:Ligands; D009154:Mutation / Q000235:genetics; D009692:Nucleic Acid Heteroduplexes / Q000235:genetics / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis; D011485:Protein Binding; D012313:RNA / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D020349:Surface Plasmon Resonance; D013698:Templates, Genetic; D013816:Thermodynamics
D017931:DNA Primers; D008024:Ligands; D009692:Nucleic Acid Heteroduplexes; D012313:RNA; D004247:DNA; D054303:HIV Reverse Transcriptase