White S A, Li H
Department of Chemistry, Bryn Mawr College, Pennsylvania 19010, USA
RNA. 1996 Mar;2(3):226-34.
Yeast ribosomal protein L32, RPL32, specifically represses splicing by binding to a purine-rich asymmetric loop adjacent to the 5' splice site of its own transcript. A potential G:U pair closes the internal loop and the goal of the present study is to understand what features of the putative G:U pair are recognized by RPL32. Two RNA oligomers containing 10 and 13 nt were annealed to form a bimolecular stem-loop-stem protein-binding site. Protein binding to each of 16 sequence variants was examined using electrophoretic bandshift and filter-binding experiments. The proteins binds to only the duplex RNA and not to the individual oligomers, and the G:U pair is critical for full-strength binding. Mutational studies show that the duplex having a G:U has the highest protein affinity (Kd = 10 nM), followed by RNAs bearing G:A, C:C, U:A, U:C, or G:G. Duplexes containing the other possible pairs bind very weakly and Watson-Crick pairing does not favor protein binding. The G of the G:U is required for strong protein binding, but replacement by inosine reduces binding only modestly. Therefore, the minor groove guanine amino group is not a key protein recognition element. Both nucleotides of the pair influence the binding strength, but their contributions are in general not additive. These data imply that the G:U is probably paired and influences binding indirectly through its effect on the conformation of the RNA.
酵母核糖体蛋白L32（RPL32）通过与其自身转录本5'剪接位点相邻的富含嘌呤的不对称环结合，特异性地抑制剪接。一个潜在的G:U碱基对封闭了内环，本研究的目的是了解RPL32识别该假定G:U碱基对的哪些特征。将含有10个和13个核苷酸的两个RNA寡聚物退火，形成一个双分子茎环 - 茎蛋白结合位点。使用电泳迁移率变动分析和滤膜结合实验检测蛋白质与16个序列变体中每一个的结合情况。该蛋白质仅与双链RNA结合，而不与单个寡聚物结合，并且G:U碱基对对于高强度结合至关重要。突变研究表明，具有G:U的双链体具有最高的蛋白质亲和力（Kd = 10 nM），其次是含有G:A、C:C、U:A、U:C或G:G的RNA。含有其他可能碱基对的双链体结合非常弱，并且沃森 - 克里克配对不利于蛋白质结合。G:U中的G对于强蛋白质结合是必需的，但用次黄嘌呤替代仅适度降低结合。因此，小沟鸟嘌呤氨基不是关键的蛋白质识别元件。该碱基对的两个核苷酸都影响结合强度，但它们的贡献通常不是相加的。这些数据表明，G:U可能是配对的，并通过其对RNA构象的影响间接影响结合。
