Luo Y, Shuman S
Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021.
J Biol Chem. 1993 Oct 5;268(28):21253-62.
Vaccinia virus capping enzyme, a heterodimer of 95-kDa and 33-kDa subunits, modifies the 5' RNA end and also acts as a transcription termination factor during synthesis of viral early mRNAs. Termination occurs in response to a specific signal, UUUUUNU, in the nascent RNA chain. We now report that purified capping enzyme binds to defined RNAs in solution to form complexes that are stable during native gel electrophoresis. Multiple enzyme molecules can bind to a single RNA. No particular 5' end structure is required for RNA binding, suggesting that the observed protein-RNA interaction is unrelated to the triphosphatase, guanylyltransferase, or methyltransferase functions of capping enzyme. Although binding does not require a UUUUUNU element in the RNA, complex formation is competed preferentially by poly(U) compared to poly(C). Capping enzyme binds to the synthetic 30-mer homopolymers to form a single protein-RNA complex; affinity for U-30 is 10-fold higher than for A-30. The sites of protein-RNA contact, as detected by UV cross-linking, are located predominantly within the 95-kDa capping enzyme subunit, which is itself sufficient to bind and cross-link to RNA in the absence of the small subunit.
痘苗病毒加帽酶是一种由95 kDa和33 kDa亚基组成的异二聚体，它修饰RNA的5'末端，并且在病毒早期mRNA合成过程中还充当转录终止因子。终止反应是对新生RNA链中的特定信号UUUUUNU作出的响应。我们现在报告，纯化的加帽酶在溶液中与特定RNA结合形成复合物，这些复合物在非变性凝胶电泳过程中是稳定的。多个酶分子可以与单个RNA结合。RNA结合不需要特定的5'末端结构，这表明观察到的蛋白质-RNA相互作用与加帽酶的三磷酸酶、鸟苷酸转移酶或甲基转移酶功能无关。虽然结合不需要RNA中有UUUUUNU元件，但与聚（C）相比，聚（U）能更有效地竞争复合物的形成。加帽酶与合成的30聚体同聚物结合形成单一的蛋白质-RNA复合物；对U-30的亲和力比对A-30高10倍。通过紫外线交联检测到的蛋白质-RNA接触位点主要位于95 kDa的加帽酶亚基内，在没有小亚基的情况下，该亚基本身就足以与RNA结合并发生交联。
