Lin Yuan, Kielkopf Clara L
Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland 21205, USA.
Biochemistry. 2008 May 20;47(20):5503-14. doi: 10.1021/bi7022392. Epub 2008 Apr 25.
A pseudouridine-modified region of the U2 small nuclear (sn)RNA anneals with the intronic branchpoint sequence and positions a bulged adenosine to serve as the nucleophile in the first chemical step of pre-mRNA splicing. We have determined three X-ray structures of RNA oligonucleotides containing the pseudouridylated U2 snRNA and the branchpoint consensus sequences. The expected adenosine branchpoint is extrahelical in a 1.65 A resolution structure containing the mammalian consensus sequence variant and in a 2.10 A resolution structure containing a shortened Saccharomyces cerevisiae consensus sequence. The adenosine adjacent to the expected branchpoint is extrahelical in a third structure, which contains the intact yeast consensus sequence at 1.57 A resolution. The hydration and base stacking interactions mediated by the U2 snRNA pseudouridines correlate with the identity of the unpaired adenosine. The expected adenosine bulge is associated with a well-stacked pseudouridine, which is linked via an ordered water molecule to a neighboring nucleotide. In contrast, the bulge of the adjacent adenosine shifts the base stacking and disrupts the water-mediated interactions of the pseudouridine. These structural differences may contribute to the ability of the pseudouridine modification to promote the bulged conformation of the branch site adenosine and to enhance catalysis by snRNAs. Furthermore, iodide binding sites are identified adjacent to the unconventional bulged adenosine, and the structure of the mammalian consensus sequence variant provides a high-resolution view of a hydrated magnesium ion bound in a similar manner to a divalent cation binding site of the group II intron.
U2小核（sn）RNA的假尿苷修饰区域与内含子分支点序列退火，并定位一个凸起的腺苷，使其在mRNA前体剪接的第一步化学反应中作为亲核试剂。我们已经确定了三种RNA寡核苷酸的X射线结构，这些寡核苷酸包含假尿苷化的U2 snRNA和分支点共有序列。在分辨率为1.65 Å的包含哺乳动物共有序列变体的结构以及分辨率为2.10 Å的包含缩短的酿酒酵母共有序列的结构中，预期的腺苷分支点处于螺旋外状态。在第三种结构中，与预期分支点相邻的腺苷处于螺旋外状态，该结构在分辨率为1.57 Å时包含完整的酵母共有序列。由U2 snRNA假尿苷介导的水合作用和碱基堆积相互作用与未配对腺苷的身份相关。预期的腺苷凸起与一个堆积良好的假尿苷相关，该假尿苷通过一个有序水分子与相邻核苷酸相连。相比之下，相邻腺苷的凸起改变了碱基堆积并破坏了假尿苷的水介导相互作用。这些结构差异可能有助于假尿苷修饰促进分支位点腺苷的凸起构象并增强snRNA催化作用的能力。此外，在非常规凸起腺苷附近鉴定出碘离子结合位点，并且哺乳动物共有序列变体的结构提供了一个高分辨率视图，显示一个水合镁离子以类似于II组内含子二价阳离子结合位点的方式结合。
