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Dynamic architecture of the RNA polymerase I transcription initiation machinery

Dynamic architecture of the RNA polymerase I transcription initiation machinery

Objective

RNA polymerase (Pol) I and Pol III synthesize non-translated RNAs including rRNA, 5S RNA, and tRNAs for ribosome assembly and protein synthesis. Pol I and Pol III transcription initiation machineries are carefully regulated in healthy cells, while misregulation of Pol I and Pol III transcription is observed in a variety of cancers. We propose to study the structure and dynamics of the Pol I transcription initiation machinery in a broad and interdisciplinary analysis that combines an integrated structural biology approach with in vitro and in vivo functional analysis, proteomics approaches, integrative modelling and fluorescence microscopy studies. The project builds on our on-going efforts in studying Pol III transcription and first results in the analysis of Pol I transcription, where crystals of the 14-subunit Pol I enzyme diffracting to at least 3.5 Å resolution have been obtained. We will determine the crystal structure of Pol I as second eukaryotic DNA-dependent RNA polymerase structure, and we will also co-crystallize Pol I with nucleic acids and Pol I-specific general transcription factors. A particular focus of the project will be the structural and functional characterization of the Pol I transcription pre-initiation complex (Pol I PIC) that is considerably smaller and compacter as the Pol II PIC. Structural data combined with results from chemical cross-linking and biochemical interaction assays will be used for integrative modeling of the structure and dynamics of Pol I PICs. Fluorescence (cross-) correlation spectroscopy (FCS/FCCS) will monitor Pol I PIC assembly in vivo. Structural information will direct the site-specific fluorescence labeling of Pol I PIC components to monitor conformational changes during Pol I PIC assembly and transcription in vitro. Our results will provide comprehensive, structural, and mechanistic insights into Pol I transcription and the possible role of Pol I in cancer.
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Principal Investigator

Christoph Müller (Dr.)

Host institution

EUROPEAN MOLECULAR BIOLOGY LABORATORY

Address

Meyerhofstrasse 1
69117 Heidelberg

Germany

Activity type

Higher or Secondary Education Establishments

EU Contribution

€ 2 459 090

Principal Investigator

Christoph Müller (Dr.)

Administrative Contact

Jillian Rowe (Ms.)

Beneficiaries (1)

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EUROPEAN MOLECULAR BIOLOGY LABORATORY

Germany

EU Contribution

€ 2 459 090

Project information

Grant agreement ID: 340964

Status

Closed project

  • Start date

    1 February 2014

  • End date

    31 January 2019

Funded under:

FP7-IDEAS-ERC

  • Overall budget:

    € 2 459 090

  • EU contribution

    € 2 459 090

Hosted by:

EUROPEAN MOLECULAR BIOLOGY LABORATORY

Germany