A Mediator-responsive form of metazoan RNA polymerase II

Abstract

RNA polymerase II (Pol II), whose 12 subunits are conserved across eukaryotes, is at the heart of the machinery responsible for transcription of mRNA. Although associated general transcription factors impart promoter specificity, responsiveness to gene- and tissue-selective activators additionally depends on the multiprotein Mediator coactivator complex. We have isolated from tissue extracts a distinct and abundant mammalian Pol II subpopulation that contains an additional tightly associated polypeptide, Gdown1. Our results establish that Gdown1-containing Pol II, designated Pol II(G), is selectively dependent on and responsive to Mediator. Thus, in an in vitro assay with general transcription factors, Pol II lacking Gdown1 displays unfettered levels of activator-dependent transcription in the presence or absence of Mediator. In contrast, Pol II(G) is dramatically less efficient in responding to activators in the absence of Mediator yet is highly and efficiently responsive to activators in the presence of Mediator. Our results reveal a transcriptional control mechanism in which Mediator-dependent regulation is enforced by means of Gdown1, which likely restricts Pol II function only to be reversed by Mediator.

Fig. 1.

Polypeptide composition and transcription elongation activities of two forms of Pol II. (A) Polypeptide composition after separation on UNO-Q HPLC, resolution on 4–20% SDS/PAGE (NuPAGE), and staining with Coomassie blue. Pol II subunits are numbered according to their yeast Pol II homologues. Identities of subunits RPB3–RPB12 were established by MS analysis. An additional stoichiometric 43-kDa polypeptide is present in Pol II(G). (B) Transcription elongation activities in a tailed template assay (39). Both early-arrested and read-through transcripts are indicated. Each reaction contained 1 μg of enzyme, a nonsaturating level that yields an activity in the linear range of the assay (Fig. 6, which is published as supporting information on the PNAS web site).

Fig. 2.

Comparison of Gdown1 and GRINL1A CTU polypeptide sequences. (A) Alignment of peptide sequences A and B (determined by Edman degradation) with GRINL1A CTU polypeptide sequences sharing common domains. Sequences were predicted from human cDNAs with GenBank accession numbers as follows: Gdown1, AF326773; Gdown2, AK074767 translated from frame 3; Gdown6, AY353061 translated from frame 1; Gcom1, AY207007. (B) Alignment of Gdown1 sequences from various species with sequences of polypeptides A and B. Protein sequences were from the Protein Information Resource (PIR) database as follows: Q96JB7, human; Q5REC6, orangutan; Q6P6I6, mouse; Q91XQ4, rat; Q9CXJ7, mouse; Q5U282, frog. The bovine sequence was deduced from partial cDNA sequences from PIR BF042463 and The Institute for Genomic Research sequences TC281745 and TC266399.

Fig. 3.

Tight association of Gdown1 with reconstituted Pol II(G). A mixture of bovine Pol II, bovine Pol II(G), and rhGdown1 was subjected to chromatography on UNO-Q, and the derived, fully reconstituted Pol II(G) was then subjected to size exclusion chromatography in the presence of 2.0 M urea and analyzed by SDS/PAGE and Coomassie staining. M represents marker proteins of 193, 103, 60, 42, and 28 kDa. Rpb1–3, native calf thymus Gdown1, and rhGdown1 are indicated.

Fig. 4.

Mediator is selectively required for activator-dependent transcription by Gdown1-containing Pol II. Transcription was assayed in a completely defined system containing GTFs, PC4, either complete TRAP/Mediator or PC2/Mediator as indicated, either HNF-4 or GAL4-AH and corresponding activator-binding DNA templates as indicated, and either natural [Pol II, Pol II(G)] or reconstituted [rec Pol II(G)] forms of Pol II as indicated. (A) HNF-4- and TRAP/Mediator-dependent transcription with Pol II and Pol II(G). (B) GAL4-AH- and TRAP/Mediator-dependent transcription with Pol II and Pol II(G). (C) HNF-4- and PC2/Mediator-dependent transcription with Pol II and Pol II(G). (D) HNF-4- and TRAP/Mediator-dependent transcription with reconstituted Pol II(G). Reconstituted Pol II(G) in D was generated either by adding a saturating amount of Gdown1 to a mixture of bovine Pol II and Pol II(G) followed by purification on UNO-Q HPLC (lanes 1 and 2) or by direct addition of Gdown1 to a reaction containing purified Pol II (lanes 7 and 8).

Fig. 5.

Model for Mediator-dependent transcription activation by Gdown1-containing Pol II. The model emphasizes formation of an activator–Mediator–Pol II(G)–GTF promoter complex in which formation of a transcriptionally competent Gdown1-free Pol II depends on the presence of the Mediator. The model allows for formation of this complex by divergent pathways, e.g., either by prior formation of an inactive activator-associated PIC followed by Mediator recruitment (Top Left) or by formation of an activator–Mediator–promoter complex followed by recruitment of Pol II(G) and GTFs (Top Right). Although the fate of Gdown1 after Pol II activation is unknown, it may, as indicated, be released for reassociation with free Pol II and subsequent reentry to the scaffold complex for Mediator-dependent reinitiation.