The nonphosphorylated form of RNA polymerase II preferentially associates with the preinitiation complex.

Abstract

The two forms of RNA polymerase H that exist in vivo, phosphorylated (HO) and nonphosphorylated (HA), were purified to apparent homogeneity from HeLa cells. The nonphosphorylated form preferentially binds to the preinitiation complex. RNA polymerase H in the complex was converted by a cellular protein kinase to the phosphorylated form.Purified RNA polymerase II cannot accurately initiate transcription from class II promoters in vitro unless it is supplemented with general transcription initiation factors (1-3). Seven human general transcription factors (TFIIA, -IIB, -IID, -lIE, -IIF, -IIG, and -IIH) that, together with RNA polymerase II are sufficient for specific transcription, have been identified (O.F. and D.R., unpublished results).Despite progress in the purification of the general transcription factors, determination of their individual activities and contributions to the formation of a transcriptioncompetent complex remains obscure. Complicating these analyses is the existence in eukaryotic cells of two different forms of RNA polymerase II, IIA and IIO, which differ in the level of phosphorylation of a highly conserved heptapeptide repeat present at the carboxyl terminus of the largest subunit (carboxyl-terminal domain; CTD) (4-6). The heptapeptide repeat is essential for viability (7-10); nevertheless, a third species ofRNA polymerase II lacking the CTD (IIB) has been observed in vitro (11)(12)(13)(14). Photoaffinity labeling experiments demonstrated that nascent RNA transcripts crosslink almost exclusively to the phosphorylated IIO form in vivo and in vitro (5,15,16), suggesting that it is the IIO polymerase that elongates RNA chains. Monoclonal antibodies against the nonphosphorylated IIA form inhibited specific transcription initiation (17, 18), suggesting that RNA polymerase IIA was more active than IIO during specific transcription initiation in vitro. Therefore, the CTD phosphorylation state may regulate the transition from initiation to elongation (19).We have purified human RNA polymerases IIO and IIA to apparent homogeneity and analyzed their roles in transcription. We demonstrate that the IIA form associates preferentially with the preinitiation complex where it is then converted by a cellular protein kinase to the phosphorylated 11O form. MATERIALS AND METHODSTranscription Factors, Transcription Reaction Mixtures, and DNA Binding Assays. Transcription reactions and DNA binding assays were performed as described (20). Transcription factor IIA (TFIIA) (P. Cortes and D.R., unpublished data), TFIIB (22), TFIIE (23), and TFIIF (24) were purified from HeLa cell nuclear extracts as described. TFIID was purified to homogeneity as described (20) Purification of the Phosphorylated (HO) and Nonphosphorylated (HA) Forms of RNA Polymerase H. RNA polymerase II was purified from HeLa cell nuclear pellets (7.5 x 1010 cells). Enzyme solubilization and chromatography on DE-52 were as described (26). Active fractions ofthe DE-52 column, between 0.2 and 0.3 M salt, were pooled (109 mg of protein, 170 ml...

Transcriptional control and the role of silencers in transcriptional regulation in eukaryotes

“…Phosphorylation of the CTD allows the removal of TFIIB, TFIIE and TFIIH (promoter clearance) [30,48,49], which in turn permits RNA pol IIo (phosphorylated form of RNA pol II) [49,50] to commence elongation [42]. Once elongation has been terminated, RNA pol IIo detaches from the DNA and, before it can re-enter initiation, the CTD must be dephosphorylated [47,51]. Assembly of the GTFs has been extensively reviewed in [30,49,52].…”

“…TFIIH has a kinase activity which can phosphorylate the CTD [57], and it appears that mediator confers a 30-50-fold stimulation of this TFIIH-dependent phosphorylation [44,59]. Finally, RNA pol IIo dephosphorylation has been attributed to the holoenzyme complex [47,51,60].…”

Modulation of TFIIH‐associated kinase activity by complex formation and its relationship with CTD phosphorylation of RNA polymerase II

“…These results indicate the special roles of the CTD in transcription and, ultimately, in ensuring cell viability. In fact, it has been reported that PolII possessing the hyperphosphorylated CTD of the largest subunit (IIo form) does not participate in the transcription PIC (Lu et al 1991), while actively transcribing PolII contains the IIo form of the largest subunit (Weeks et al 1993;O'Brien et al 1994). Recently, it was reported that the phosphorylated CTD has a role in recruiting the mRNA capping enzyme to the nascent transcript and that mRNA capping occurs soon after promoter clearance (Coppola et al 1983;Cho et al 1997;McCracken et al 1997a,b;Yue et al 1997).…”

“…The CTD has been shown to interact with various factors which form the preinitiation multi-polypeptide complex, including the TATAbinding protein (TBP) and the PolII/SRB (suppressor of RNA polymerase B)/Med (mediator found as a component of yeast holoenzyme) complex (Usheva et al 1992; Thompson et al 1993;Myers et al 1998). The unphosphorylated form of PolII (Pol IIa) is preferentially recruited into the PIC reconstituted from puri®ed general transcription factors (Lu et al 1991). CTD phosphorylation may initially occur between transcription initiation and the transition from initiation to elongation, converting PolII to the phosphorylated form (Pol IIo) (O'Brien et al 1994;Svejstrup et al 1996;Ohkuma 1997).…”

Anti-RNA polymerase antibodies in systemic sclerosis (SSc): association with anti-topoisomerase I antibodies and identification of autoreactive subunits of RNA polymerase II

“…Unlike RNAPs I and III, the largest subunit of RNAP II has a characteristic carboxyl-terminal repeat domain [17], which is the site of multiple phosphorylation/ dephosphorylation reactions which control intraconversion of the two forms [18,19]. The largest subunit of RNAP IIO (designated IIo) is conformationally distinct from that of RNAP IIA (subunit IIa) [20], and the two forms of the enzyme are active at different stages of the transcription process, sometimes in a site-specific manner [18,[21][22][23][24]. It is therefore possible that autoantibodies could recognize either one or both types of RNAP II.…”