Involvement of mRNA capping enzyme in transcription initiation

MRNA的封顶发生在转录启动后不久和其他加工事件之前。这几个mRNA加工因子通过与RNA聚合酶II(CTD)的C-末端结构域结合而与转录耦合。MRNA封顶酶直接与磷酸化的CTD结合，并催化共转录帽子的形成。然而，封闭酶参与PolII转录的确切机制尚不清楚。(1)为了研究封闭酶在PolII转录中的作用，我们利用固定化DNA模板建立了体外转录/封盖体系。将固定化模板与HeLa核抽提物孵育，在启动子区域形成起始复合体(PIC)。在洗涤去除游离蛋白质后，PIC在3‘-O-甲基CTP存在下进行伸长反应，以便在第一个C残基停止伸长复合体。利用该系统，我们发现在腺病毒主要晚期启动子的情况下，封帽发生在18-19个核苷酸的RNA长度。(2)为了研究封帽酶与PolII的功能相互作用，我们用各种CTD激酶对高纯度的小牛胸腺PolII进行封盖实验。用硼酸盐亲和电泳法(BAGE)分离封端RNA，评价封端效率。我们发现，非磷酸化的PolII刺激了大约2.5倍的封顶活性。此外，CAK磷酸化polII的封帽活性是未磷酸化polII的1.8倍。这些结果表明POLII CTD的CAK磷酸化对于封闭酶的激活是重要的，PolII的其他部分也可能参与了这一激活。

The mRNA capping occurs soon after transcription initiation and before other processing events. The several mRNA processing factors are coupled to transcription through binding to the C-terminal domain of RNA polymerase II (CTD). mRNA-capping enzyme directly binds to the phosphorylated CTD, and catalyzes the co-transcriptional cap formation. However, the precise mechanism of the involvement of capping enzyme in pol II transcription has been unclear.(1) To examine the involvement of capping enzymes in pol II transcription, we developed in vitro transcription/capping system using immobilized DNA template. Immobilized templates were incubated with HeLa nuclear extract to form initiation complex (PICs) on promoter region. After washing to remove unbound proteins, PICs were subjected to elongation reaction in the presence of 3'-O-methly CTP in order to stall elongation complex at the first C residue. Using this system, we found that capping occurs in 18-19 nt RNA length in the case of adenovirus major late promoter.(2) To investigate the functional interaction of capping enzyme and pol II, we subjected highly purified calf thymus pol II to capping assay with various kinds of CTD kinases. Capped RNAs were separated by Boronate-affinity PAGE (BAGE) to evaluate capping efficiency. We found that non-phosphorylated pol II stimulated capping activity about 2.5-fold. Furthermore, CAK phosphorylated pol II increased in capping activity about 1.8-fold compared with non-phosphorylated pol II. These results suggest that the CAK phosphorylation of pol II CTD is important for activation of capping enzyme and other portion of pol II also may involve in this activation.