Architecture of RNA Polymerase II and Implications for the Transcription Mechanism

Cramer, Patrick; Bushnell, David; Fu, Jiyang; Gnatt, Averell; Maier-Davis, Barbara; Thompson, Nancy E.; Burgess, Richard R.; Edwards, A.M.; David, Paul R.; Kornberg, Roger D.

doi:10.1126/science.288.5466.640

Cited by 558 publications

(465 citation statements)

References 78 publications

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“…Traditional structure determination methods other than electron microscopy, such as NMR and X-ray crystallography, can generally not be applied to the structure of these large macromolecular complexes, with a few spectacular exceptions such as the ribosome (Yusupov et al, 2001) and RNA polymerase (Cramer et al, 2000). Many biologically highly relevant macromolecules in the cell can only be found in small copy numbers, making biochemical puriWcation procedures diYcult and usually not eYcient enough to produce complexes in the amount and homogeneity needed for structural studies by X-ray crystallography.…”

Section: Discussionmentioning

confidence: 99%

Advantages of CCD detectors for de novo three-dimensional structure determination in single-particle electron microscopy

Sander

Golas

Stark

2005

Journal of Structural Biology

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For three-dimensional (3D) structure determination of large macromolecular complexes, single-particle electron cryomicroscopy is considered the method of choice. Within this Weld, structure determination de novo, as opposed to reWnement of known structures, still presents a major challenge, especially for macromolecules without point-group symmetry. This is primarily because of technical issues: one of these is poor image contrast, and another is the often low particle concentration and sample heterogeneity imposed by the practical limits of biochemical puriWcation. In this work, we tested a state-of-the art 4k £ 4k charge-coupled device (CCD) detector (TVIPS TemCam-F415) to see whether or not it can contribute to improving the image features that are especially important for structure determination de novo. The present study is therefore focused on a comparison of Wlm and CCD detector in the acquisition of images in the low-to-medium (»10-25 Å) resolution range using a 200 kV electron microscope equipped with Weld emission gun. For comparison, biological specimens and radiation-insensitive carbon layers were imaged under various conditions to test the image phase transmission, spatial signal-to-noise ratio, visual image quality and power-spectral signal decay for the complete imageprocessing chain. At all settings of the camera, the phase transmission and spectral signal-to-noise ratio were signiWcantly better on CCD than on Wlm in the low-to-medium resolution range. Thus, the number of particle images needed for initial structure determination is reduced and the overall quality of the initial computed 3D models is improved. However, at high resolution, Wlm is still signiWcantly better than the CCD camera: without binning of the CCD camera and at a magniWcation of 70k£, Wlm is better beyond 21 Å resolution. With 4-fold binning of the CCD camera and at very high magniWcation (>300k£) Wlm is still superior beyond 7 Å resolution. 

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Section: Discussionmentioning

confidence: 99%

Advantages of CCD detectors for de novo three-dimensional structure determination in single-particle electron microscopy

Sander

Golas

Stark

2005

Journal of Structural Biology

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“…At the center of the transcription machinery, the 12-subunit Pol II enzyme contains two large subunits, Rpb1 and Rpb2, that form a central cleft for nucleic acid entry and contain the enzyme active site [16][17][18] . The remaining 10 smaller subunits (Rpb3-Rpb12) surround these two largest subunits to provide structural support and to regulate Pol II enzymatic activity.…”

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The positions of TFIIF and TFIIE in the RNA polymerase II transcription preinitiation complex

Chen

Warfield

Hahn

2007

Nat Struct Mol Biol

159

187

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The non-natural photoreactive amino acid p-Benzoyl-L-Phenylalanine (Bpa) was incorporated into the RNA polymerase (Pol) II surface surrounding the central cleft formed by the Rpb1 and Rpb2 subunits. Photocrosslinking of Preinitiation Complexes (PICs) with these Pol II derivatives and hydroxyl radical cleavage assays revealed that the TFIIF dimerization domain interacts with the Rpb2 lobe and protrusion domains adjacent to Rpb9 while TFIIE crosslinks to the Rpb1 clamp domain on the opposite side of the Pol II central cleft. Mutations in the Rpb2 lobe and protrusion domains were found to alter both Pol II-TFIIF binding and the transcription start site, a phenotype associated with mutations in TFIIF, Rpb9, and TFIIB. In combination with previous biochemical and structural studies, these new findings illuminate the structural organization of the PIC and reveal a network of protein-protein interactions involved in transcription start site selection.The earliest step in transcription initiation is recruitment of Pol II and the general transcription factors to form the Preinitiation Complex (PIC) 1 . Upon addition of ATP, the PIC transitions to the Open Complex state, resulting in separation of the DNA strands surrounding the transcription start site and insertion of the template strand into the active center of Pol II. Next, Pol II must locate the transcription start site, which in S. cerevisiae, can be over 60 base pairs distant from the initial site of DNA strand separation 2 . Mutations in the general factor TFIIF and in the B-finger domain of the general factor TFIIB can alter the transcription start site, suggesting these two factors are involved in start site recognition 3-7 . Consistent with this role, crystallography and protein-protein crosslinking in the PIC have positioned both of these factors within the Pol II active site cleft 8-10 . Additionally, mutations in the Pol II subunit Rpb9 have been found to alter the transcription start site, but it remains unclear how this subunit, which is located distant from the Pol II active site, participates in start site selection 11,12 .Initiation of RNA synthesis begins with DNA-NTP base pairing, phosphodiester bond formation, and translocation of the DNA-RNA hybrid within the active center of Pol II. After synthesis of 8−12 bases of RNA, Pol II escapes from the promoter into a stable elongation state 13-15 . The nucleation of transcription factors at the promoter and the structural transition into the open and elongation complexes involves a complex set of protein-protein and protein-DNA interactions. Elucidating these molecular interactions within the transcription machinery Phone: 206 667 5261 Fax 206 667 6497 shahn@fhcrc.org. AUTHOR CONTRIBUTIONS L.W. modified the non-natural amino acid incorporation system and performed the experiment in Figure 1. H-T.C. performed and designed the remaining experiments. S.H. supervised the study. H-T.C. and S.H. wrote the manuscript.Publisher's Disclaimer: This PDF receipt will only be used as the basis for generating ...

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“…Structure determination has revealed a core devoted to nucleic acid polymerization and an elaborate superstructure for the recognition of diverse promoter sequences and also for regulation (1)(2)(3). The proportion of the structure devoted to these different tasks may be gauged by comparison with the single-subunit viral RNA polymerases (4,5), which are approximately one-fifth of the mass of the multisubunit enzymes and transcribe only one or a small number of promoters, with rudimentary requirements for control.…”

mentioning

confidence: 99%

Diffusion of nucleoside triphosphates and role of the entry site to the RNA polymerase II active center

Batada

Westover

Bushnell

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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Nucleoside triphosphates (NTPs) diffuse to the active center of RNA polymerase II through a funnel-shaped opening that narrows to a negatively charged pore. Computer simulation shows that the funnel and pore reduce the rate of diffusion by a factor of Ϸ2 ؋ 10 ؊7 . The resulting limitation on the rate of RNA synthesis under conditions of low NTP concentration may be overcome by NTP binding to an entry site adjacent to the active center. Binding to the entry site greatly enhances the lifetime of an NTP in the active center region, and it prevents ''backtracking'' and the consequent occlusion of the active site.computation ͉ electrostatics ͉ transcription T he RNA polymerases responsible for cellular gene transcription are large multiprotein complexes. Structure determination has revealed a core devoted to nucleic acid polymerization and an elaborate superstructure for the recognition of diverse promoter sequences and also for regulation (1-3). The proportion of the structure devoted to these different tasks may be gauged by comparison with the single-subunit viral RNA polymerases (4, 5), which are approximately one-fifth of the mass of the multisubunit enzymes and transcribe only one or a small number of promoters, with rudimentary requirements for control.Among the many structural accoutrements of the multisubunit enzymes, one that has attracted recent attention is an undercarriage that serves an important purpose in transcript elongation but that also imposes an apparent limitation on the enzyme mechanism. This undercarriage harbors a funnel-shaped opening, narrowing to a pore beneath the active center ( Fig. 1) through which the 3Ј end of the RNA is extruded during ''backtracking'' (retrograde motion of the enzyme on the DNA template) and through which substrate nucleoside triphosphates (NTPs) must enter for addition to the 3Ј end during RNA synthesis. Backtracking is crucial for recovery from pausing, for exposing DNA damage for repair, and possibly for proofreading the nascent transcript (reviewed in ref. 6). Pausing is of particular interest for its regulatory role. Recovery from pausing generally requires transcript cleavage in the polymerase active center, induced by the general transcription factors SII (TFIIS) in eukaryotes and GreA͞B in bacteria. Defects in pausing and recovery underlie human diseases such as Cockayne syndrome, Wolf-Hirschhorn syndrome, and von Hippel-Lindau cancerpredisposition syndrome.The polymerase funnel and pore facilitate pausing and recovery. A backtracked transcript is sequestered, possibly with its 3Ј end bound to a site of nucleotide interaction in the funnel at approximately nine residues from the active center (7). The transcript is protected from nucleases, accessible only to TFIIS or GreA͞B, which bind to the surface of the funnel and possess a long, slender protrusion that is capable of reaching up through the pore to the active center (8, 9). The transcript and protrusion can occupy the pore and funnel simultaneously. The protrusion is thought to supply a Mg ion that is...

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Architecture of RNA Polymerase II and Implications for the Transcription Mechanism

Abstract: This copy is for your personal, non-commercial

Cited by 558 publications

References 78 publications

Advantages of CCD detectors for de novo three-dimensional structure determination in single-particle electron microscopy

Advantages of CCD detectors for de novo three-dimensional structure determination in single-particle electron microscopy

The positions of TFIIF and TFIIE in the RNA polymerase II transcription preinitiation complex

Diffusion of nucleoside triphosphates and role of the entry site to the RNA polymerase II active center

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