Abstract
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.
Highlights
The largest subunit of R N A polymerase 11( R N A P II) Pro-Ser, at its COOH terminus which is tandemly repeated26, contains a remarkable region of tandem heptapeptide 44, and 52 times in yeast, Drosophila, and mammalian cells, repeats of the consensus sequence ’&r-Ser-Pro-Thr-Ser- respectively (Allison etal., 1985; Corden etal., 1985)
The analysis of preinitiation complexes assembledon the DHFR promoter indicates that RNAP IIA readily assembles into functional preinitiation complexes in contrast to the inefficient assembly of RNAP 110.transcript elongation is catalyzed by RNAP I10 as demonstrated by the photoactivated cross-linkingof nascent DHFR transcripts to subunit 110
This paper addresses the involvement of the CTD in transcription from the murine DHFR promoter using transcription reactions dependent on the addition of exogenousRNAP 11.The reconstituted transcription system allows for the direct assessment of the role of the CTD in DHFR transcription since the absence/presence and stateof phosphorylation of the CTD can be controlled.The studies presented here indicate that theCTD plays a direct role in therecruitment of RNAP I1 to the DHFR promoter
Summary
Materials-Ultrapure nucleotides were purchased from Pharmacia LKB Biotechnology Inc. Radiolabeled ribonucleotides[CY-~~PIC(3T0P00 Ci/mmol)and [y-32PlATP (3000 Ci/mmol) werepurchased from Amersham Corp. In Vitro Banscription Reactions-Reconstituted transcription reactions contained either 6 or 12 pl of DE0.25 (as indicated in the figure legends), 30-50 ng of Spl, 2.540milliunits of RNAP I1 (as indicated), 24 m~ Hepes-KOH, pH7.9,6 m~ MgCl,, 60 m~ KCl, 0.12 m~ EDTA, 0.3 rn D m , 0.12 m~ phenylmethylsulfonyl fluoride, 12% glycerol, 25 p~. To make 32P-labeledRNAP 110, 3ZP-labeledRNAP IIA was incubated with excess cold ATP and a partially purified fraction ofCTD kinase (Chesnut et al, 1992) Both 32P-labeleRdNAPs IIA and I10were purified as previously described(Chestnut et al, 1992),dialyzed into Buffer D and assayed for promoter-independenttranscriptional activity (Kim and Dahmus, 1988). Preinitiation complexes were assembled on the DHFR promoter with 32P-labeledRNAP IIA, 110, or a mixture of RNAPs IIA and I10 (milliunits indicated in the figure legends) in a 3 x transcription reaction (total volume was 60 p1) byincubation for 45 min a t 24 "C in theabsence of ribonucleotides. Column fractions (50 PI) were treated with DNase I (0.50unit) for 30 min on ice and analyzed by electrophoresis on a 5% polyacrylamide-SDSgel by silver staining and autoradiography
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