Kang M E, Dahmus M E
Section of Molecular and Cellular Biology, University of California, Davis 95616.
J Biol Chem. 1993 Nov 25;268(33):25033-40.
The largest subunit of RNA polymerase II (RNAP II) contains a remarkable region of tandem heptapeptide repeats of the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser at its carboxyl terminus. This COOH-terminal domain (CTD) is unphosphorylated in RNAP IIA, extensively phosphorylated in RNAP IIO, and absent in RNAP IIB. The reversible phosphorylation of the CTD has been proposed to be integral to each cycle of transcription from the adenovirus-2 major late promoter. The adenovirus-2 major late promoter, however, may not be a good paradigm for the study of CTD function because in vitro transcription from this promoter is not dependent on the CTD. Previous studies suggest that transcription from the murine dihydrofolate reductase (DHFR) promoter requires the CTD. In an effort to investigate the role of the CTD and its phosphorylation, a RNAP II-dependent reconstituted transcription system specific for the DHFR promoter was established. In this reconstituted system, RNAP IIA, but not RNAP IIB, can transcribe from the DHFR promoter. Furthermore, RNAP IIB does not compete with RNAP IIA for preinitiation complex assembly. These results suggest that the CTD plays a critical role in the recruitment of RNAP II to the DHFR promoter. The analysis of preinitiation complexes assembled on the DHFR promoter indicates that RNAP IIA readily assembles into functional preinitiation complexes in contrast to the inefficient assembly of RNAP IIO. However, transcript elongation is catalyzed by RNAP IIO as demonstrated by the photoactivated cross-linking of nascent DHFR transcripts to subunit IIo. These results indicate that transcription from the DHFR promoter involves the reversible phosphorylation of the CTD and support the idea that RNAPs IIA and IIO have essential but distinct functions.
RNA聚合酶II（RNAP II）的最大亚基在其羧基末端含有一段由共有序列Tyr-Ser-Pro-Thr-Ser-Pro-Ser组成的显著的串联七肽重复区域。这个羧基末端结构域（CTD）在RNAP IIA中未被磷酸化，在RNAP IIO中被广泛磷酸化，而在RNAP IIB中不存在。CTD的可逆磷酸化被认为是腺病毒2型主要晚期启动子转录每个周期所必需的。然而，腺病毒2型主要晚期启动子可能不是研究CTD功能的良好范例，因为从该启动子进行的体外转录不依赖于CTD。先前的研究表明，从小鼠二氢叶酸还原酶（DHFR）启动子进行的转录需要CTD。为了研究CTD及其磷酸化的作用，建立了一个依赖于RNAP II的针对DHFR启动子的重组转录系统。在这个重组系统中，RNAP IIA而非RNAP IIB能够从DHFR启动子进行转录。此外，RNAP IIB不会与RNAP IIA竞争预起始复合物的组装。这些结果表明，CTD在将RNAP II招募到DHFR启动子的过程中起着关键作用。对在DHFR启动子上组装的预起始复合物的分析表明，与RNAP IIO低效组装形成对比的是，RNAP IIA能够轻易组装成功能性的预起始复合物。然而，新生DHFR转录本与亚基IIo的光活化交联表明，转录延伸是由RNAP IIO催化的。这些结果表明，从DHFR启动子进行的转录涉及CTD的可逆磷酸化，并支持了RNAPs IIA和IIO具有重要但不同功能的观点。
