Probing the kinetics of formation of the bacteriophage MS2 translational operator complex: Identification of a protein conformer unable to bind RNA
Probing the kinetics of formation of the bacteriophage MS2 translational operator complex: Identification of a protein conformer unable to bind RNA
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DOI：
10.1006/jmbi.2000.4355
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发表时间：
2001-02-02
影响因子：
5.6
通讯作者：
Stockley, PG
中科院分区：
文献类型：
--
作者：
Lago, H;Parrott, AM;Stockley, PG
我们研究了噬菌体MS2外壳蛋白亚基与包含天然翻译操作位点的合成RNA片段之间复合物形成的动力学，或已知与蛋白质上相同位点结合的三个不同RNA适体家族的共识序列。用停止流动荧光光谱分析反应，用荧光腺苷类似物2'-脱氧，2-氨基嘌呤特异性取代蛋白质的固有色氨酸荧光或RNA片段的信号。观察到的动力学与被监测的荧光团或其在配合物中的位置无关，表明数据报告了在配合物形成过程中发生的全局事件。竞争分析表明，正在形成的复合物由一个外壳蛋白二聚体和一个RNA分子组成。结合反应至少是双相的。较快的阶段占振幅的80- 85%，主要是扩散驱动的rna -蛋白质相互作用(k(1)近似于2 × 10（9) M-1 s(-1)）。正向反应的盐依赖性和低亲和RNA片段上速率的相似性与静电导向主导的扩散控制步骤一致。慢相与反应物浓度无关，似乎与RNA结合前外壳蛋白亚基的异构化相对应（k（iso）近似于0.23 s(-1)）。对外壳蛋白突变体（Pro78Asn）的测量表明，这一阶段不是由于该残基的顺式异构化。在rna -蛋白复合物形成过程中，蛋白质配体的构象变化可能在触发衣壳自组装中起作用，并讨论了这一模型。(C) 2001学术出版社。
We have investigated the kinetics of complex formation between bacteriophage MS2 coat protein subunits and synthetic RNA fragments encompassing the natural translational operator site, or the consensus sequences of three distinct RNA aptamer families, which are known to bind to the same site on the protein. Reactions were assayed using stopped-flow fluorescence spectroscopy and either the intrinsic tryptophan fluorescence of the protein or the signals from RNA fragments site-specifically substituted with the fluorescent adenosine analogue 2'-deoxy, 2-aminopurine. The kinetics observed were independent of the fluorophore being monitored or its position within the complex, indicating that the data report global events occurring during complex formation. Competition assays show that the complex being formed consists of a single coat protein dimer and one RNA molecule. The binding reaction is at least biphasic. The faster phase, constituting 80-85 % of the amplitude, is a largely diffusion driven RNA-protein interaction (k(1) approximate to 2 x 10(9) M-1 s(-1)). The salt dependence of the forward reaction and the similarities of the on-rates of lower-affinity RNA fragments are consistent with a diffusion-controlled step dominated by electrostatic steering. The slower phase is independent of reactant concentration, and appears to correspond to isomerisation of the coat protein subunit(s) prior to RNA binding (k(iso) approximate to 0.23 s(-1)). Measurements with a coat protein mutant (Pro78Asn) show that this phase is not due to cis-tuans isomerisation at this residue. The conformational changes in the protein ligand during formation of an RNA-protein complex might play a role in the triggering of capsid self-assembly and a model for this is discussed. (C) 2001 Academic Press.