Title

Binding of Human Immunodeficiency Virus Type 1 Nucleocapsid Protein to Ψ-RNA-SL3

Author(s)/Creator(s)

Michael F. Shubsda, Syracuse University
Christopher A. Kirk, Syracuse University
Jerry Goodisman, Syracuse UniversityFollow
James C. Dabrowiak, Syracuse UniversityFollow

Document Type

Article

Date

7-17-2000

Keywords

Nucleocapsid, Electrophoresis, Protein-RNA complex

Disciplines

Chemistry

Description/Abstract

The interaction of the nucleocapsid protein NCp7, from the pNL4-3 isolate of HIV-1, with Ψ-RNA-SL3, with the sequence 5’-GGACUAGCGGAGGCUAGUCC, was studied using non-denaturing gel electrophoresis. Two kinds of experiments were performed, using buffered solutions of radiolabeled RNA and unlabeled protein. In the ‘dilution’ experiments, the total RNA concentration, R_T, was varied for a series of solutions, but kept equal to the total protein concentration, P_T. In the ‘titration’ experiments, solutions having R_T constant but with varying P_T were analyzed. The solutions were electrophoresed and the autoradiographic spot intensities, proportional to the amounts of the different species present, were measured. The intensities were fit to a number of equilibrium models, differing in species stoichiometries, by finding the best values of the binding constants. It was shown that NCp7 protein and SL3 RNA combine to form at least two complexes. When P_T is below approximately 10 μM, a complex that contains two RNAs and one protein forms. Increasing P_T to approximately 100 μM causes the 2:1 complex to oligomerize, forming a species having eight RNAs and four proteins. For the dilution experiments, run at 5 ̊C at an ionic strength of 31 mM, we found K₁ for the 2:1 complex is ̴ 10¹¹ M^-2 and K₂ for the 8:4 complex is ̴ 10¹⁶ M^-3. The titration experiments returned K₁ ̴ 10⁷ M^-2 (poorly determined) and K₁ ̴ 10¹⁹ M^-3. The analysis was complicated by the loss of RNA at higher protein concentrations, due to formation of an insoluble species containing both RNA and protein, which does not enter the gel. Correcting for this changes the calculated values of equilibrium constants, but not the molecularities determined by our analysis. The observation that a small complex can oligomerize to form a larger species is consistent with the fact that NCp7 organizes and condenses the genome in the virus particle.

Additional Information

Copyright 2000 Biophysical Chemistry. This article may be downloaded for personal use only. Any other use requires prior permission of the author and Biophysical Chemistry.

The article may be found at

doi: 10.1016/S0301- 4622(00)00188-5

Recommended Citation

Shubsda, Michael F.; Kirk, Christopher A.; Goodisman, Jerry; and Dabrowiak, James C., "Binding of Human Immunodeficiency Virus Type 1 Nucleocapsid Protein to Ψ-RNA-SL3" (2000). Chemistry - All Scholarship. 84.
https://surface.syr.edu/che/84

Source

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