Shubsda M F, Kirk C A, Goodisman J, Dabrowiak J C
Department of Chemistry, Center for Science and Technology, Syracuse University, NY 13224-4100, USA.
Biophys Chem. 2000 Oct 30;87(2-3):149-65. doi: 10.1016/s0301-4622(00)00188-5.
The interaction of the nucleocapsid protein NCp7, from the pNL4-3 isolate of HIV-1, with psi-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 microM, a complex that contains two RNAs and one protein forms. Increasing PT to approximately 100 microM causes the 2:1 complex to oligomerize, forming a species having eight RNAs and four proteins. For the dilution experiments, run at 5 degrees C at an ionic strength of 31 mM, we found K1 for the 2:1 complex is approximately 10(11) M(-2) and K2 for the 8:4 complex is approximately 10(16) M(-3). The titration experiments returned K1 approximately 10(7) M(-2) (poorly determined) and K2 approximately 10(19) 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.
利用非变性凝胶电泳研究了来自HIV-1的pNL4-3分离株的核衣壳蛋白NCp7与序列为5'-GGACUAGCGGAGGCUAGUCC的ψ-RNA-SL3之间的相互作用。进行了两类实验，分别使用放射性标记RNA和未标记蛋白的缓冲溶液。在“稀释”实验中，一系列溶液的总RNA浓度RT发生变化，但保持与总蛋白浓度PT相等。在“滴定”实验中，分析了RT恒定但PT不同的溶液。对这些溶液进行电泳，并测量与存在的不同物种数量成比例的放射自显影片斑点强度。通过找到结合常数的最佳值，将强度拟合到一些在物种化学计量上不同的平衡模型。结果表明，NCp7蛋白和SL3 RNA结合形成至少两种复合物。当PT低于约10微摩尔时，形成一种包含两个RNA和一个蛋白的复合物。将PT增加到约100微摩尔会使2:1复合物寡聚化，形成一种具有八个RNA和四个蛋白的物种。对于在5℃、离子强度为31 mM下进行的稀释实验，我们发现2:1复合物的K1约为10(11) M(-2)，8:4复合物的K2约为10(16) M(-3)。滴定实验得到的K1约为10(7) M(-2)（测定结果不佳），K2约为10(19) M(-3)。由于形成了一种包含RNA和蛋白的不溶性物种（该物种不进入凝胶），在较高蛋白浓度下RNA会损失，这使得分析变得复杂。对此进行校正会改变平衡常数的计算值，但不会改变我们分析确定的分子组成。小复合物可以寡聚化形成更大物种的观察结果与NCp7在病毒颗粒中组织和浓缩基因组这一事实相符。
