Crosslinking-MS analysis reveals RNA polymerase I domain architecture and basis of rRNA cleavage

Jennebach, Stefan; Herzog, Franz; Aebersold, Ruedi; Cramer, Patrick

doi:10.1093/nar/gks220

Cited by 66 publications

(72 citation statements)

References 40 publications

(85 reference statements)

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“…DNA (42,(44)(45)(46), and all are located proximal to or above the DNA binding channel (13,15,16,35,39,(47)(48)(49). We propose that TFE-like factors operate via two mechanisms: (i) they act as allosteric effectors by favoring RNAP clamp opening and (ii) simultaneously tether the nontemplate strand in the PIC.…”

Section: Dna Melting Correlates With Clamp Openingmentioning

confidence: 99%

TFE and Spt4/5 open and close the RNA polymerase clamp during the transcription cycle

Schulz

Gietl

Smollett

et al. 2016

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

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Transcription is an intrinsically dynamic process and requires the coordinated interplay of RNA polymerases (RNAPs) with nucleic acids and transcription factors. Classical structural biology techniques have revealed detailed snapshots of a subset of conformational states of the RNAP as they exist in crystals. A detailed view of the conformational space sampled by the RNAP and the molecular mechanisms of the basal transcription factors E (TFE) and Spt4/5 through conformational constraints has remained elusive. We monitored the conformational changes of the flexible clamp of the RNAP by combining a fluorescently labeled recombinant 12-subunit RNAP system with single-molecule FRET measurements. We measured and compared the distances across the DNA binding channel of the archaeal RNAP. Our results show that the transition of the closed to the open initiation complex, which occurs concomitant with DNA melting, is coordinated with an opening of the RNAP clamp that is stimulated by TFE. We show that the clamp in elongation complexes is modulated by the nontemplate strand and by the processivity factor Spt4/5, both of which stimulate transcription processivity. Taken together, our results reveal an intricate network of interactions within transcription complexes between RNAP, transcription factors, and nucleic acids that allosterically modulate the RNAP during the transcription cycle.

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Section: Dna Melting Correlates With Clamp Openingmentioning

confidence: 99%

TFE and Spt4/5 open and close the RNA polymerase clamp during the transcription cycle

Schulz

Gietl

Smollett

et al. 2016

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

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“…We then analysed this complex by XL-MS. The power and reliability of XL-MS was recently exemplified by a high agreement between Pol I models derived from XL-MS 13 and subsequently from X-ray analysis 14 .…”

Section: Resultsmentioning

confidence: 99%

“…0.9 mg purified ITC (1.2 mg ml À 1 ) was incubated with an eightfold molar excess of DNA-RNA scaffold and crosslinked with 0.6 mM isotope-labeled disuccimidyl suberate (DSS-d0/d12, Creative Molecules Inc.) as described 13 . Crosslinked protein was digested, and the crosslinked peptides were enriched, analysed by liquid chromatography coupled to tandem mass spectrometer (Orbitrap Elite), and spectra were searched by the xQuest software 31,32 .…”

Section: Methodsmentioning

confidence: 99%

Conserved architecture of the core RNA polymerase II initiation complex

et al. 2014

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During transcription initiation at promoters of protein-coding genes, RNA polymerase (Pol) II assembles with TBP, TFIIB and TFIIF into a conserved core initiation complex that recruits additional factors. The core complex stabilizes open DNA and initiates RNA synthesis, and it is conserved in the Pol I and Pol III transcription systems. Here, we derive the domain architecture of the yeast core pol II initiation complex during transcription initiation. The yeast complex resembles the human initiation complex and reveals that the TFIIF Tfg2 winged helix domain swings over promoter DNA. An 'arm' and a 'charged helix' in TFIIF function in transcription start site selection and initial RNA synthesis, respectively, and apparently extend into the active centre cleft. Our model provides the basis for further structure-function analysis of the entire transcription initiation complex.

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“…A two-fold non-crystallographic symmetry (NCS) axis was detected by calculation of a self-rotation function. Molecular replacement using as search models Pol II, a Pol I homology model 11 or a Pol I electron microscopic envelope 9 failed. We could, however, use anomalous diffraction from intrinsically bound zinc ions for phasing.…”

Section: Research Article Methodsmentioning

confidence: 99%

“…The mobile A49 tWH domain (Fig. 1b) may close over nucleic acids in the cleft 11 to increase transcription processivity 9,21 . The A49-A34.5 subcomplex is related to the Pol II initiation factor TFIIF and to the Pol III subcomplex C37-C53, which contain similar dimerization domains located at corresponding positions [22][23][24] .…”

Section: Composite Active Centrementioning

confidence: 99%

RNA polymerase I structure and transcription regulation

Engel

Sainsbury

Cheung

et al. 2013

Nature

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199

305

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Transcription of ribosomal RNA by RNA polymerase (Pol) I initiates ribosome biogenesis and regulates eukaryotic cell growth. The crystal structure of Pol I fromthe yeast Saccharomyces cerevisiae at 2.8A˚ resolution reveals all 14 subunits of the 590-kilodalton enzyme, and shows differences to Pol II. An ‘expander’ element occupies the DNA template site and stabilizes an expanded active centre cleft with an unwound bridge helix. A ‘connector’ element invades the cleft of an adjacent polymerase and stabilizes an inactive polymerase dimer. The connector and expander must detach during Pol I activation to enable transcription initiation and cleft contraction by convergent movement of the polymerase ‘core’ and ‘shelf’ modules. Conversion between an inactive expanded and an active contracted polymerase state may generally underlie transcription. Regulatory factors can modulate the core–shelf interface that includes a ‘composite’ active site for RNA chain initiation, elongation, proofreading and termination

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Crosslinking-MS analysis reveals RNA polymerase I domain architecture and basis of rRNA cleavage

Cited by 66 publications

References 40 publications

TFE and Spt4/5 open and close the RNA polymerase clamp during the transcription cycle

TFE and Spt4/5 open and close the RNA polymerase clamp during the transcription cycle

Conserved architecture of the core RNA polymerase II initiation complex

RNA polymerase I structure and transcription regulation

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