Involvement of mRNA capping enzyme in transcription initiation

mRNA盖帽发生在转录起始后不久和其他加工事件之前。几种mRNA加工因子通过与RNA聚合酶II （CTD）的c端结构域结合而偶联转录。mRNA-capping酶直接与磷酸化的CTD结合，催化共转录帽的形成。然而，盖帽酶参与pol II转录的确切机制尚不清楚。(1)为了研究capping酶在pol II转录中的作用，我们利用固定化DNA模板构建了体外转录/capping系统。固定化模板与HeLa核提取物孵育，在启动子区域形成起始复合物（PICs）。在洗涤去除未结合蛋白后，PICs在3'- o - methyl CTP的存在下进行延伸反应，以阻止第一个C残基的延伸络合物。利用该系统，我们发现在腺病毒主要晚期启动子的情况下，封顶发生在18-19 nt RNA长度上。(2)为了研究封盖酶与pol II的功能相互作用，我们用不同的CTD激酶对高纯度的小牛胸腺pol II进行了封盖实验。用硼酸盐亲和PAGE （BAGE）技术对封盖rna进行分离，评价封盖效率。我们发现非磷酸化的pol II刺激了约2.5倍的capping活性。此外，与未磷酸化的pol II相比，CAK磷酸化的pol II的capping活性提高了约1.8倍。这些结果表明，CAK磷酸化的pol II CTD对capping酶的激活是重要的，并且pol II的其他部分也可能参与了这种激活。

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.