Spicer E, Schwarzbauer J, Craven G R
Nucleic Acids Res. 1977 Feb;4(2):491-9. doi: 10.1093/nar/4.2.491.
E. coli ribosomal proteins are retained by nitrocellulose filters. In contrast, 16S RNA passes through nitrocellulose filters. We have found that specific protein-RNA complexes involving single proteins also pass through nitrocellulose filters. Thus, by utilizing radioactively labeled r-proteins, nitrocellulose filtration can be used to study directly and sensitively the stoichiometry of r-protein-RNA association. The filtration process maintains near equilibrium conditions, making it applicable to weak as well as strong protein-RNA associations. We have used nitrocellulose filtration to obtain saturation binding curves for the association of proteins S4, S7, S8 and S20 with 16S RNA. In each case, the stoichiometry of binding was one mole of protein or less per mole of RNA. The stoichiometry of protein S8 binding to 16S RNA measured by filtration is comparable to that observed by sucrose gradient centrifugation. Association constants for the binding of proteins S4, S8 and S20 to 16S RNA have been determined by analysis of the saturation binding curves and were found to range from .3-6 X 10(7)M-1.
大肠杆菌核糖体蛋白能被硝酸纤维素滤膜截留。相比之下，16S RNA能透过硝酸纤维素滤膜。我们发现，涉及单个蛋白的特定蛋白-RNA复合物也能透过硝酸纤维素滤膜。因此，通过使用放射性标记的核糖体蛋白，硝酸纤维素过滤可用于直接且灵敏地研究核糖体蛋白与RNA结合的化学计量关系。过滤过程维持着接近平衡的条件，使其适用于弱的以及强的蛋白-RNA结合。我们已使用硝酸纤维素过滤来获得蛋白质S4、S7、S8和S20与16S RNA结合的饱和结合曲线。在每种情况下，结合的化学计量关系为每摩尔RNA结合一摩尔或更少的蛋白质。通过过滤测得的蛋白质S8与16S RNA结合的化学计量关系与通过蔗糖梯度离心观察到的结果相当。通过对饱和结合曲线的分析，已确定了蛋白质S4、S8和S20与16S RNA结合的结合常数，发现其范围为0.3 - 6×10⁷M⁻¹。
