Tycon Michael A, Daddysman Matthew K, Fecko Christopher J
Department of Chemistry, University of North Carolina at Chapel Hill , Chapel Hill, North Carolina 27599-3290, United States.
J Phys Chem B. 2014 Jan 16;118(2):423-33. doi: 10.1021/jp4082933. Epub 2013 Dec 31.
Nearly all cellular processes are enacted by multi-subunit protein complexes, yet the assembly mechanism of most complexes is not well understood. The anthropomorphism "protein recruitment" that is used to describe the concerted binding of proteins to accomplish a specific function conceals significant uncertainty about the underlying physical phenomena and chemical interactions governing the formation of macromolecular complexes. We address this deficiency by investigating the diffusion dynamics of two RNA polymerase II subunits, Rpb3 and Rpb9, in regions of live Drosophila cell nuclei that are devoid of chromatin binding sites. Using FRAP microscopy, we demonstrate that both unengaged subunits are incorporated into a broad distribution of complexes, with sizes ranging from free (unincorporated) proteins to those that have been predicted for fully assembled gene transcription units. In live cells, Rpb3 exhibits regions of stability at both size extremes connected by a continuous distribution of complexes. Corresponding measurements on cellular extracts reveal a distribution that retains peaks at the extremes but not in between, suggesting that partially assembled complexes are less stable. We propose that the broad distribution of macromolecular species allows for mechanistic flexibility in the assembly of transcription complexes.
几乎所有的细胞过程都是由多亚基蛋白质复合物执行的，然而大多数复合物的组装机制尚未得到很好的理解。用于描述蛋白质协同结合以完成特定功能的拟人化说法“蛋白质招募”掩盖了关于控制大分子复合物形成的潜在物理现象和化学相互作用的重大不确定性。我们通过研究果蝇活细胞核中两个RNA聚合酶II亚基Rpb3和Rpb9在没有染色质结合位点的区域的扩散动力学来解决这一不足。使用荧光恢复后光漂白（FRAP）显微镜，我们证明这两个未结合的亚基都被纳入了广泛分布的复合物中，其大小范围从游离（未结合）蛋白质到那些已被预测为完全组装好的基因转录单元的复合物。在活细胞中，Rpb3在大小两端都表现出稳定区域，由连续分布的复合物连接。对细胞提取物的相应测量揭示了一种分布，其在两端保留峰值，但中间没有，这表明部分组装的复合物不太稳定。我们提出，大分子物种的广泛分布允许转录复合物组装过程中的机制灵活性。
