Studies on Primer Binding of HIV-1 Reverse Transcriptase Using a Fluorescent Probe

• Published in: Journal of molecular biology Vol. 236; no. 2; pp. 469 - 479
• Main Authors: Delahunty, Martha D., Wilson, Samuel H., Karpel, Richard L.
• Format: Journal Article
• Language: English
• Published: Oxford Elsevier Ltd 18.02.1994; Elsevier
• Subjects: Adenosine Triphosphate - analogs & derivatives; Adenosine Triphosphate - metabolism; AIDS/HIV; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Enzymes and enzyme inhibitors; fluorescence; Fluorescent Dyes; Fundamental and applied biological sciences. Psychology; HIV Reverse Transcriptase; HIV-1 - enzymology; human immunodeficiency virus 1; Kinetics; nucleotide analog; primer binding site; reverse transcriptase; RNA, Transfer, Lys - metabolism; RNA-Directed DNA Polymerase - metabolism; Spectrometry, Fluorescence; Transferases; primer binding site; reverse transcriptase; fluorescence; nucleotide analog; RNA-directed DNA polymerase; HIV-1 virus; Enzyme; Transferases; Retroviridae; Primer; Molecular interaction; Binding site; Virus; Substrate; Nucleotidyltransferases; Analog; Nucleotide; Lentivirinae; Affinity; Human immunodeficiency virus
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1006/jmbi.1994.1158

The fluorescent nucleotide analog, 2′,3′-trinitrophenyladenosine-5′-triphosphate (TNP-ATP), was utilized to quantify the affinities of human immunodeficiency virus-1 reverse transcriptase (HIV-1 RT) for its substrates. Interaction of this probe with the enzyme brings about a twofold increase in the magnitude of fluoresence emission from the probe, and a blue-shift in wavelength maximum, from 561 to 553 nm. TNP-ATP binds HIV-1 RT with a dissociation constant of 21 μM. The presence of millimolar levels of deoxynucleoside triphosphates or micromolar levels of an oligonucleotide primer analogue, p(dT) 12-18, suppressed this enhancement of fluoresce. The fact that inhibition was achieved with much lower levels of primer than of dNTPs suggests that TNP-ATP is a probe for the binding site of primer of the enzyme, rather than that of deoxynucleoside triphosphate. In support of this, the effect of TNP-ATP on the kinetics of DNA synthesis catalyzed by the enzyme indicated that the probe is a competitive inhibitor with respect to template-primer. The ability of primers and primer analogs to reverse the fluoresence enhancement was determined, and the corresponding affinities of these compounds for reverse for reverse transcriptase were calculated. The affinity increased with primer length, increasing more than 50-fold from a span of 5 to 15 nucleotide residues. The interaction of polydeoxynucleotides was consistent with a model in which the enzyme bound at adjacent internal sites of about 15 residues in length. Several mammalian and bacterial transfer RNA primers were tested, including the natural primer, tRNA Lys 3. The affinities were found to be between 0·55 and 1·2 μM, with no obvious selectivity for the natural primer, which had a K d of 0·79 μM. These results are discussed within the context of data for HIV-1 RT obtained by other methodologies.