Architecture of initiation-competent 12-subunit RNA polymerase II

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: 9.81). 07/2003; 100(12):6964-8. DOI: 10.1073/pnas.1030608100
Source: PubMed

ABSTRACT 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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    • "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 · 33.12 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 · 6.79 Impact Factor
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    • ": RNA polymerase II structure. (a) Side view of the core Pol II crystal structure containing all twelve subunits and displaying the RNA exit channel (bold arrow) and the positioning of the CTD adapted from Armache et al. [71]. Cartoon in the upper right displays the color coding for the Pol II subunits used in the crystal structure. "
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    ABSTRACT: The C-terminal domain (CTD) of RNA polymerase II (Pol II) consists of conserved heptapeptide repeats that function as a binding platform for different protein complexes involved in transcription, RNA processing, export, and chromatin remodeling. The CTD repeats are subject to sequential waves of posttranslational modifications during specific stages of the transcription cycle. These patterned modifications have led to the postulation of the "CTD code" hypothesis, where stage-specific patterns define a spatiotemporal code that is recognized by the appropriate interacting partners. Here, we highlight the role of CTD modifications in directing transcription initiation, elongation, and termination. We examine the major readers, writers, and erasers of the CTD code and examine the relevance of describing patterns of posttranslational modifications as a "code." Finally, we discuss major questions regarding the function of the newly discovered CTD modifications and the fundamental insights into transcription regulation that will necessarily emerge upon addressing those challenges.
    02/2012; 2012:347214. DOI:10.1155/2012/347214
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