Nucleocapsid, Electrophoresis, Protein-RNA complex
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, RT, was varied for a series of solutions, but kept equal to the total protein concentration, PT. In the ‘titration’ experiments, solutions having RT constant but with varying PT 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 PT is below approximately 10 μM, a complex that contains two RNAs and one protein forms. Increasing PT 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.
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 - Faculty Scholarship. 84.
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