Schoumacher F, Gagey M J, Maira M, Stussi-Garaud C, Godefroy-Colburn T
Institut de Biologie Moléculaire des Plantes, Université Louis Pasteur, Strasbourg, France.
FEBS Lett. 1992 Aug 24;308(3):231-4. doi: 10.1016/0014-5793(92)81281-p.
The movement protein of alfalfa mosaic virus was expressed in Escherichia coli and purified by cation exchange chromatography. The purified protein bound single-stranded RNA cooperatively in a biphasic manner. At protein saturation, RNA/protein complexes (designated 'primary complexes') were detected by a nitrocellulose-retention assay within 1 min of mixing, both at 4 and 22 degrees C. In contrast, an incubation of 30 min at 22 degrees C was necessary to obtain electrophoretically retarded complexes ('stabilized complexes'), containing a large number of protein molecules bound stably to each molecule of RNA. Stabilization did not take place at 4 degrees C. The rate of formation of the primary complexes was strongly dependent on protein concentration, and thus appeared limited by a bimolecular interaction. In contrast, the rate of stabilization was independent of protein concentration, suggesting that this process consisted of a rearrangement of the primary complexes without binding of additional protein molecules. In agreement with this suggestion, the amount of complexed RNA at equilibrium was the same when assayed by nitrocellulose retention and by electrophoretic retardation. The possibility that these peculiar kinetics could be caused by the presence of Tween 20 in the incubation media is discussed.
苜蓿花叶病毒的运动蛋白在大肠杆菌中表达，并通过阳离子交换色谱法进行纯化。纯化后的蛋白以双相方式协同结合单链RNA。在蛋白质饱和时，在4℃和22℃下混合1分钟内，通过硝酸纤维素保留试验检测到RNA/蛋白质复合物（称为“初级复合物”）。相比之下，在22℃下孵育30分钟才能获得电泳迁移率减慢的复合物（“稳定复合物”），其中每个RNA分子都稳定结合有大量蛋白质分子。在4℃下不会发生稳定化。初级复合物的形成速率强烈依赖于蛋白质浓度，因此似乎受双分子相互作用的限制。相比之下，稳定化速率与蛋白质浓度无关，这表明该过程由初级复合物的重排组成，而没有额外蛋白质分子的结合。与该建议一致，通过硝酸纤维素保留和电泳迁移率测定法在平衡时检测到的复合RNA量是相同的。文中讨论了孵育介质中存在吐温20可能导致这些特殊动力学的可能性。
