DeKoster Gregory T, Delaney Kimberly J, Hall Kathleen B
Department of Biochemistry and Molecular Biophysics, Washington University Medical School. St. Louis, Missouri.
Department of Biochemistry and Molecular Biophysics, Washington University Medical School. St. Louis, Missouri.
Biophys J. 2014 Jul 1;107(1):208-19. doi: 10.1016/j.bpj.2014.05.026.
The U1A/U2B″/SNF family of small nuclear ribonucleoproteins uses a phylogenetically conserved RNA recognition motif (RRM1) to bind RNA stemloops in U1 and/or U2 small nuclear RNA (snRNA). RRMs are characterized by their α/β sandwich topology, and these RRMs use their β-sheet as the RNA binding surface. Unique to this RRM family is the tyrosine-glutamine-phenylalanine (YQF) triad of solvent-exposed residues that are displayed on the β-sheet surface; the aromatic residues form a platform for RNA nucleobases to stack. U1A, U2B″, and SNF have very different patterns of RNA binding affinity and specificity, however, so here we ask how YQF in Drosophila SNF RRM1 contributes to RNA binding, as well as to domain stability and dynamics. Thermodynamic double-mutant cycles using tyrosine and phenylalanine substitutions probe the communication between those two residues in the free and bound states of the RRM. NMR experiments follow corresponding changes in the glutamine side-chain amide in both U1A and SNF, providing a physical picture of the RRM1 β-sheet surface. NMR relaxation and dispersion experiments compare fast (picosecond to nanosecond) and intermediate (microsecond-to-millisecond) dynamics of U1A and SNF RRM1. We conclude that there is a network of amino acid interactions involving Tyr-Gln-Phe in both SNF and U1A RRM1, but whereas mutations of the Tyr-Gln-Phe triad result in small local responses in U1A, they produce extensive microsecond-to-millisecond global motions throughout SNF that alter the conformational states of the RRM.
小核糖核蛋白的U1A/U2B″/SNF家族利用系统发育保守的RNA识别基序（RRM1）结合U1和/或U2小核RNA（snRNA）中的RNA茎环。RRM以其α/β三明治拓扑结构为特征，这些RRM利用其β折叠作为RNA结合表面。该RRM家族的独特之处在于β折叠表面上暴露于溶剂中的酪氨酸 - 谷氨酰胺 - 苯丙氨酸（YQF）三联体；芳香族残基形成了RNA核碱基堆积的平台。然而，U1A、U2B″和SNF具有非常不同的RNA结合亲和力和特异性模式，因此在这里我们探讨果蝇SNF RRM1中的YQF如何促进RNA结合以及结构域的稳定性和动力学。使用酪氨酸和苯丙氨酸取代的热力学双突变循环探测RRM游离态和结合态中这两个残基之间的通讯。核磁共振实验跟踪U1A和SNF中谷氨酰胺侧链酰胺的相应变化，提供RRM1β折叠表面的物理图像。核磁共振弛豫和色散实验比较U1A和SNF RRM1的快速（皮秒到纳秒）和中间（微秒到毫秒）动力学。我们得出结论，在SNF和U1A RRM1中都存在涉及Tyr - Gln - Phe的氨基酸相互作用网络，但是虽然Tyr - Gln - Phe三联体的突变在U1A中导致小的局部响应，但它们在整个SNF中产生广泛的微秒到毫秒级全局运动，从而改变RRM的构象状态。
