Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

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

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.

Related Concept Videos

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by
Same author

Impact of Ketorolac on Opioid Consumption after Knee Arthroscopy.

Austin journal of anesthesia and analgesia·2020
Same author

Effect of Intraoperative Phenylephrine Infusion on Redistribution Hypothermia During Cesarean Delivery Under Spinal Anesthesia.

Journal of clinical anesthesia and management·2020
Same author

Maternal and procedural factors associated with estimated blood loss in second trimester surgical uterine evacuation: a retrospective cohort analysis.

International journal of obstetric anesthesia·2020
Same author

Structural and Volumetric Brain MRI Findings in Mild Traumatic Brain Injury.

AJNR. American journal of neuroradiology·2020
Same author

A simple but precise method for quantitative measurement of the quality of the laser focus in a scanning optical microscope.

Journal of microscopy·2015
Same author

A review of recent experiments on step-to-step "hand-off" of the DNA intermediates in mammalian base excision repair pathways.

Molekuliarnaia biologiia·2011

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:

Related Experiment Videos

  • 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.