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Related Experiment Videos

Studies on primer binding of HIV-1 reverse transcriptase using a fluorescent probe

M D Delahunty1, S H Wilson, R L Karpel

  • 1Department of Chemistry and Biochemistry, University of Maryland Baltimore County 21228.

Journal of Molecular Biology
|February 18, 1994
PubMed
Summary
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This study used a fluorescent probe, TNP-ATP, to measure binding affinities of HIV-1 reverse transcriptase (RT) for its substrates. Results indicate TNP-ATP primarily binds to the primer site, offering insights into enzyme-substrate interactions.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzymology

Background:

  • Human immunodeficiency virus-1 reverse transcriptase (HIV-1 RT) is a key target for antiviral therapies.
  • Understanding the substrate binding specificities of HIV-1 RT is crucial for developing effective inhibitors.
  • Fluorescent nucleotide analogs offer a sensitive method for studying enzyme-ligand interactions.

Purpose of the Study:

  • To quantify the binding affinities of HIV-1 RT for its substrates using the fluorescent analog 2',3'-trinitrophenyladenosine-5'-triphosphate (TNP-ATP).
  • To determine whether TNP-ATP preferentially binds to the deoxynucleoside triphosphate (dNTP) or primer binding site on HIV-1 RT.
  • To investigate the influence of primer length and type on binding affinity to HIV-1 RT.

Main Methods:

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  • Utilized the fluorescent nucleotide analog TNP-ATP to monitor binding to HIV-1 RT through changes in fluorescence emission.
  • Assessed the effect of dNTPs and oligonucleotide primers on TNP-ATP-induced fluorescence enhancement.
  • Determined competitive inhibition kinetics of TNP-ATP with respect to template-primer.
  • Calculated dissociation constants (Kd) for various primers and primer analogs binding to HIV-1 RT.

    • Main Results:

    • TNP-ATP binds HIV-1 RT with a dissociation constant of 21 microM and exhibits a fluorescence enhancement upon binding.
    • Fluorescence enhancement was suppressed by oligonucleotide primers at lower concentrations than dNTPs, suggesting TNP-ATP probes the primer site.
    • TNP-ATP acts as a competitive inhibitor with respect to template-primer during DNA synthesis.
    • Primer affinity increased with length, and tRNA primers bound with affinities between 0.55 and 1.2 microM.

    Conclusions:

    • TNP-ATP is a valuable fluorescent probe for studying the primer binding site of HIV-1 RT.
    • Primer length significantly influences binding affinity, supporting a model of enzyme interaction with extended primer sites.
    • HIV-1 RT shows broad affinity for various tRNA primers, not exclusively favoring the natural primer.