Lee J M, Greenleaf A L
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
J Biol Chem. 1997 Apr 25;272(17):10990-3. doi: 10.1074/jbc.272.17.10990.
Hyperphosphorylation of the C-terminal heptapeptide repeat domain (CTD) of the RNA polymerase II largest subunit has been suggested to play a key role in regulating transcription initiation and elongation. To facilitate investigating functional consequences of CTD phosphorylation we developed new templates, the double G-less cassettes, which make it possible to assay simultaneously the level of initiation and the efficiency of elongation. Using these templates, we examined the effects of yeast CTD kinase I or CTD kinase inhibitors on transcription and CTD phosphorylation in HeLa nuclear extracts. Our results showed that polymerase II elongation efficiency and CTD phosphorylation are greatly reduced by CTD kinase inhibitors, whereas both are greatly increased by CTD kinase I; in contrast, transcription initiation is much less affected. These results demonstrate that CTD kinase I modulates the elongation efficiency of RNA polymerase II and are consistent with the idea that one function of CTD phosphorylation is to promote effective production of long transcripts by stimulating the elongation efficiency of RNA polymerase II.
RNA聚合酶II最大亚基的C末端七肽重复结构域（CTD）的过度磷酸化被认为在调节转录起始和延伸中起关键作用。为了便于研究CTD磷酸化的功能后果，我们开发了新的模板——双G缺失盒，它使得同时检测起始水平和延伸效率成为可能。使用这些模板，我们研究了酵母CTD激酶I或CTD激酶抑制剂对HeLa细胞核提取物中转录和CTD磷酸化的影响。我们的结果表明，CTD激酶抑制剂可大大降低聚合酶II的延伸效率和CTD磷酸化，而CTD激酶I则可使其大大增加；相反，转录起始受影响较小。这些结果表明，CTD激酶I调节RNA聚合酶II的延伸效率，并且与CTD磷酸化的一个功能是通过刺激RNA聚合酶II的延伸效率来促进长转录本的有效产生这一观点一致。
