Zhao Ying, Baranger Anne M
Department of Chemistry, Wesleyan University, Middletown, CT 06459, USA.
J Am Chem Soc. 2003 Mar 5;125(9):2480-8. doi: 10.1021/ja021267w.
The RNA recognition motif (RRM), one of the most common RNA binding domains, contains three highly conserved aromatic amino acids that participate in stacking interactions with RNA bases. We have investigated the contribution of these highly conserved aromatic amino acids to the affinity of the complex formed between the N-terminal RRM of the U1A protein and stem loop 2 of U1 snRNA. Previously, we found that substitution of one of these conserved aromatic amino acids, Phe56, with Ala resulted in a large destabilization of the complex. Here, we have modified A6, the base in stem loop 2 RNA that stacks with Phe56, to compensate for a portion of the destabilization caused by the Phe56Ala mutation. We have designed two modified adenosines, A-3CPh and A-4CPh, in which a phenyl group is linked to the adenosine such that it may replace the phenyl group that is eliminated by the Phe56Ala mutation in the complex. We have found that incorporation of A-3CPh into stem loop 2 RNA stabilizes the complex formed with Phe56Ala by 0.6 kcal/mol, while incorporation of A-4CPh into stem loop 2 RNA stabilizes this complex by 1.8 kcal/mol. Either base modification destabilizes the wild-type complex by 0.8-0.9 kcal/mol. Experiments with other U1A mutant proteins suggest that the stabilization of the complex between the Phe56Ala U1A protein and stem loop 2 RNA is due to a specific interaction between the Phe56Ala U1A protein and A6-4CPh stem loop 2 RNA.
RNA识别基序（RRM）是最常见的RNA结合结构域之一，包含三个高度保守的芳香族氨基酸，它们参与与RNA碱基的堆积相互作用。我们研究了这些高度保守的芳香族氨基酸对U1A蛋白N端RRM与U1 snRNA茎环2形成的复合物亲和力的贡献。此前，我们发现将这些保守芳香族氨基酸之一的苯丙氨酸56（Phe56）替换为丙氨酸会导致复合物的大幅不稳定。在此，我们对茎环2 RNA中与Phe56堆积的碱基A6进行了修饰，以补偿由Phe56Ala突变引起的部分不稳定。我们设计了两种修饰的腺苷，A - 3CPh和A - 4CPh，其中一个苯基连接到腺苷上，使其可以取代复合物中因Phe56Ala突变而消除的苯基。我们发现，将A - 3CPh掺入茎环2 RNA可使与Phe56Ala形成的复合物稳定0.6千卡/摩尔，而将A - 4CPh掺入茎环2 RNA可使该复合物稳定1.8千卡/摩尔。任何一种碱基修饰都会使野生型复合物不稳定0.8 - 0.9千卡/摩尔。对其他U1A突变蛋白的实验表明，Phe56Ala U1A蛋白与茎环2 RNA之间复合物的稳定是由于Phe56Ala U1A蛋白与A6 - 4CPh茎环2 RNA之间的特异性相互作用。
