Architecture of initiation-competent 12-subunit RNA polymerase II. Proc Natl Acad Sci USA

Institute of Biochemistry and Gene Center, University of Munich, Feodor-Lynen-Strasse 25, 81377 Munich, Germany.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 07/2003; 100(12):6964-8. DOI: 10.1073/pnas.1030608100
Source: PubMed


RNA polymerase (Pol) II consists of a 10-polypeptide catalytic core and the two-subunit Rpb4/7 complex that is required for transcription initiation. Previous structures of the Pol II core revealed a "clamp," which binds the DNA template strand via three "switch regions," and a flexible "linker" to the C-terminal repeat domain (CTD). Here we derived a model of the complete Pol II by fitting structures of the core and Rpb4/7 to a 4.2-A crystallographic electron density map. Rpb4/7 protrudes from the polymerase "upstream face," on which initiation factors assemble for promoter DNA loading. Rpb7 forms a wedge between the clamp and the linker, restricting the clamp to a closed position. The wedge allosterically prevents entry of the promoter DNA duplex into the active center cleft and induces in two switch regions a conformation poised for template-strand binding. Interaction of Rpb4/7 with the linker explains Rpb4-mediated recruitment of the CTD phosphatase to the CTD during Pol II recycling. The core-Rpb7 interaction and some functions of Rpb4/7 are apparently conserved in all eukaryotic and archaeal RNA polymerases but not in the bacterial enzyme.

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    • "The Rpb4/7 stalk is easily dissociable from the 10 subunit core RNAP II in vitro (37,38). Association of the Rpb4/7 stalk with the core RNAP II ‘wedges’ the clamp to the closed conformation, resulting in a narrower central cleft of the polymerase (Figure 3B) (37,38). "
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    ABSTRACT: Spt5, a transcription elongation factor, and Rpb4, a subunit of RNA polymerase II (RNAP II) that forms a subcomplex with Rpb7, play important roles in transcription elongation and repression of transcription coupled DNA repair (TCR) in eukaryotic cells. How Spt5 physically interacts with RNAP II, and if and/or how Spt5 and Rpb4/7 coordinate to achieve the distinctive functions have been enigmatic. By site-specific incorporation of the unnatural amino acid p-benzoyl-L-phenylalanine, a photoreactive cross-linker, we mapped interactions between Spt5 and RNAP II in Saccharomyces cerevisiae. Through its KOW4-5 domains, Spt5 extensively interacts with Rpb4/7. Spt5 also interacts with Rpb1 and Rpb2, two largest subunits of RNAP II, at the clamp, protrusion and wall domains. These interactions may lock the clamp to the closed conformation and enclose the DNA being transcribed in the central cleft of RNAP II. Deletion of Spt5 KOW4-5 domains decreases transcription elongation and derepresses TCR. Our findings suggest that Spt5 is a key coordinator for holding the RNAP II complex in a closed conformation that is highly competent for transcription elongation but repressive to TCR.
    Nucleic Acids Research 05/2014; 42(11). DOI:10.1093/nar/gku333 · 9.11 Impact Factor
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    • "Binding of the 10 subunit Pol II core (Cramer et al., 2001) to the Pol II subcomplex Rpb4/7 (Armache et al., 2005) generates the complete, 12 subunit enzyme (Armache et al., 2003; Bushnell and Kornberg, 2003). Rpb4/7 binding stabilizes a closed conformation of the Pol II clamp domain, which only permits passage of singlestranded DNA to the active site (Armache et al., 2003). The complete Pol II is apparently relevant for initiation and elongation because Rpb4/7 is required for initiation in vitro (Edwards et al., 1991) and because the complete Pol II is associated with the genome in vivo (Jasiak et al., 2008). "
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    ABSTRACT: We provide here a molecular movie that captures key aspects of RNA polymerase II initiation and elongation. To create the movie, we combined structural snapshots of the initiation-elongation transition and of elongation, including nucleotide addition, translocation, pausing, proofreading, backtracking, arrest, reactivation, and inhibition. The movie reveals open questions about the mechanism of transcription and provides a useful teaching tool.
    Cell 06/2012; 149(7):1431-7. DOI:10.1016/j.cell.2012.06.006 · 32.24 Impact Factor
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    • "These unidentified densities can be better appreciated in a difference map between the Head-mPIC cryo-EM structure and the fitted Head and RNAPII modules (Figure 6A, light blue surfaces, and Figure S11, light blue mesh). We based our interpretation of these additional features in the Head-mPIC structure on information about the interaction of RNAPII with TFIIF (Chen et al., 2007; Chen et al., 2010; Eichner et al., 2010), and on the structure of the RNAPII-TFIIB complex from X-ray crystallographic studies (Armache et al., 2003; Bushnell and Kornberg, 2003; Kostrewa et al., 2009; Liu et al., 2010). "
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    ABSTRACT: Mediator, a large (21 polypeptides, MW ∼1 MDa) complex conserved throughout eukaryotes, plays an essential role in control of gene expression by conveying regulatory signals that influence the activity of the preinitiation complex. However, the precise mode of interaction between Mediator and RNA polymerase II (RNAPII), and the mechanism of regulation by Mediator remain elusive. We used cryo-electron microscopy and reconstituted in vitro transcription assays to characterize a transcriptionally-active complex including the Mediator Head module and components of a minimum preinitiation complex (RNAPII, TFIIF, TFIIB, TBP, and promoter DNA). Our results reveal how the Head interacts with RNAPII, affecting its conformation and function.
    Structure 05/2012; 20(5):899-910. DOI:10.1016/j.str.2012.02.023 · 5.62 Impact Factor
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