Institute of Structural Biology, Helmholtz Zentrum München, Ingolstädter Landstrasse 1, 85764 Neuherberg, Germany.
Nature. 2011 Jul 13;475(7356):408-11. doi: 10.1038/nature10171.
Many cellular functions involve multi-domain proteins, which are composed of structurally independent modules connected by flexible linkers. Although it is often well understood how a given domain recognizes a cognate oligonucleotide or peptide motif, the dynamic interaction of multiple domains in the recognition of these ligands remains to be characterized. Here we have studied the molecular mechanisms of the recognition of the 3'-splice-site-associated polypyrimidine tract RNA by the large subunit of the human U2 snRNP auxiliary factor (U2AF65) as a key early step in pre-mRNA splicing. We show that the tandem RNA recognition motif domains of U2AF65 adopt two remarkably distinct domain arrangements in the absence or presence of a strong (that is, high affinity) polypyrimidine tract. Recognition of sequence variations in the polypyrimidine tract RNA involves a population shift between these closed and open conformations. The equilibrium between the two conformations functions as a molecular rheostat that quantitatively correlates the natural variations in polypyrimidine tract nucleotide composition, length and functional strength to the efficiency to recruit U2 snRNP to the intron during spliceosome assembly. Mutations that shift the conformational equilibrium without directly affecting RNA binding modulate splicing activity accordingly. Similar mechanisms of cooperative multi-domain conformational selection may operate more generally in the recognition of degenerate nucleotide or amino acid motifs by multi-domain proteins.
许多细胞功能都涉及多结构域蛋白，这些蛋白由通过柔性连接体连接的结构独立模块组成。虽然人们通常很清楚给定的结构域如何识别同源的寡核苷酸或肽基序，但多个结构域在识别这些配体时的动态相互作用仍有待研究。在这里，我们研究了人类 U2 snRNP 辅助因子（U2AF65）大亚基识别 3'-剪接位点相关多嘧啶tract RNA 的分子机制，这是 pre-mRNA 剪接的关键早期步骤。我们表明，在不存在或存在强（即高亲和力）多嘧啶 tract 的情况下，U2AF65 的串联 RNA 识别结构域采用两种截然不同的结构域排列。多嘧啶 tract RNA 序列变异的识别涉及这两种封闭和开放构象之间的群体转移。这两种构象之间的平衡作为分子变阻器，将多嘧啶 tract 核苷酸组成、长度和功能强度的自然变化与剪接体组装过程中 U2 snRNP 招募到内含子的效率定量相关。不直接影响 RNA 结合的改变构象平衡的突变会相应地调节剪接活性。类似的合作多结构域构象选择机制可能更普遍地存在于多结构域蛋白识别简并核苷酸或氨基酸基序的过程中。
