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Affinity of reverse transcriptase for some polynucleotide inhibitors
Archives of Biochemistry and Biophysics
|February 1, 1984
Summary
Avian myeloblastosis virus reverse transcriptase affinity for polynucleotides is influenced by modifications and molecular weight. Enzyme-polynucleotide complex stability depends on salt concentration but not Mg+2.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Avian myeloblastosis virus reverse transcriptase is a key enzyme in retroviral replication.
- Polynucleotides, particularly polyuridylic acid ((U)n), can interact with and inhibit reverse transcriptase.
- Understanding these interactions is crucial for developing antiviral therapies.
Purpose of the Study:
- To quantify the relative binding affinities of avian myeloblastosis virus reverse transcriptase for (U)n and its analogs.
- To investigate the impact of polynucleotide structure and molecular weight on enzyme affinity.
- To determine the influence of Mg+2 and salt concentrations on enzyme-polynucleotide complex stability.
Main Methods:
- Electron spin resonance (ESR) spectroscopy was employed to measure binding affinities.
- A quantitative competition assay was utilized, involving a spin-labeled polynucleotide probe.
- Relative affinities were determined by calculating the concentration of competing polynucleotides required for enzyme displacement.
Main Results:
- Relative affinities varied significantly based on (U)n modifications and molecular weight, e.g., K(dUfl)n = 1.6, K(dT)n = 20, K(U)80 > 167.
- Enzyme binding was observed in both the presence and absence of Mg+2.
- Increasing KCl concentration from 0.04M to 0.5M completely dissociated the polynucleotide-enzyme complex.
Conclusions:
- The affinity of (U)n for avian myeloblastosis virus reverse transcriptase is sensitive to both the chemical modifications of the polynucleotide matrix and its molecular weight.
- The stability of the enzyme-polynucleotide complex is highly dependent on ionic strength, with high salt concentrations disrupting binding.
- These findings provide insights into the molecular basis of reverse transcriptase inhibition by polynucleotides.
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