NMR structure and dynamics of an RNA motif common to the spliceosome branch-point helix and the RNA-binding site for phage GA coat protein.
NMR structure and dynamics of an RNA motif common to the spliceosome branch-point helix and the RNA-binding site for phage GA coat protein.
复制标题
剪接体分支点螺旋和噬菌体 GA 外壳蛋白的 RNA 结合位点共有的 RNA 基序的 NMR 结构和动力学。
DOI：
10.1021/bi981558a
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Nikonowicz,EP
中科院分区：
文献类型：
--
作者：
Smith,JS;Nikonowicz,EP
组成剪接体分支点螺旋和噬菌体 GA 外壳蛋白结合位点的 RNA 分子共享一个二级结构基序，其中两个连续的腺嘌呤残基占据与单个尿苷相对的链，从而有可能形成两个不同的 A·U 碱基对之一，同时使另一个腺嘌呤不配对或凸出。在前体 mRNA 的内含子剪接过程中，凸出的腺嘌呤的 2'-OH 参与 5'-外显子的酯交换反应，并形成套索中间体的分支点残基。任一腺嘌呤均可充当哺乳动物中的分支点残基，但 3'-近端腺嘌呤优先充当分支点残基。当与噬菌体 GA 外壳蛋白结合时，凸出的腺嘌呤环从螺旋中伸出并占据蛋白质表面的结合袋，形成用于噬菌体组装的成核复合物。外壳蛋白可以在任一位置结合带有凸出腺嘌呤的螺旋，但 3'-近端位点的结合亲和力更大。我们研究了 21 个核苷酸发夹中的 RNA 基序，该发夹含有 GA 外壳蛋白结合位点，其四个核苷酸环已被来自相关噬菌体 Ms2 的更稳定的环取代。使用异核核磁共振波谱，我们确定了该发夹结构的整体精度为 2.0 Å。两个腺嘌呤碱基堆积成螺旋，虽然所有可用的 NOE 和偶联常数数据都与两个可能的 A·U 碱基对一致，但涉及 5'-近端腺嘌呤的碱基对似乎是主要构象。 3'-近端凸出的腺嘌呤在异常高的 pH 下质子化，为了解释这一点，我们提出了一个模型，其中质子化的腺嘌呤通过与 A·U 碱基对的尿苷 O2 上的氢键来稳定。凸出的腺嘌呤的2'-OH采用规则的A型螺旋几何形状，这表明为了参与剪接反应，活性剪接体中分支点螺旋的构象可能会与此处描述的构象发生变化。因此，虽然剪接体和噬菌体 GA 的腺嘌呤位点偏好可能是由于蛋白质因素，但优选的腺嘌呤在游离 RNA 中易于发生参与活性复合物形成的构象重排。
The RNA molecules that make up the spliceosome branch-point helix and the binding site for phage GA coat protein share a secondary structure motif in which two consecutive adenine residues occupy the strand opposite a single uridine, creating the potential to form one of two different A·U base pairs while leaving the other adenine unpaired or bulged. During the splicing of introns out of pre-mRNA, the 2‘-OH of the bulged adenine participates in the transesterification reaction at the 5‘-exon and forms the branch-point residue of the lariat intermediate. Either adenine may act as the branch-point residue in mammals, but the 3‘-proximal adenine does so preferentially. When bound to phage GA coat protein, the bulged adenine loops out of the helix and occupies a binding pocket on the surface of the protein, forming a nucleation complex for phage assembly. The coat protein can bind helices with bulged adenines at either position, but the 3‘-proximal site binds with greater affinity. We have studied this RNA motif in a 21 nucleotide hairpin containing a GA coat protein-binding site whose four nucleotide loop has been replaced by a more stable loop from the related phage Ms2. Using heteronuclear NMR spectroscopy, we have determined the structure of this hairpin to an overall precision of 2.0 Å. Both adenine bases stack into the helix, and while all available NOE and coupling constant data are consistent with both possible A·U base pairs, the base pair involving the 5‘-proximal adenine appears to be the major conformation. The 3‘-proximal bulged adenine protonates at unusually high pH, and to account for this, we propose a model in which the protonated adenine is stabilized by a hydrogen bond to the uridine O2 of the A·U base pair. The 2‘-OH of the bulged adenine adopts a regular A-form helical geometry, suggesting that in order to participate in the splicing reaction, the conformation of the branch-point helix in the active spliceosome may change from the conformation described here. Thus, while the adenine site preferences of the spliceosome and of phage GA may be due to protein factors, the preferred adenine is predisposed in the free RNA to conformational rearrangement involved in formation of the active complexes.