eprintid: 27386 rev_number: 16 eprint_status: archive userid: 4797 dir: disk0/00/02/73/86 datestamp: 2019-11-20 14:01:59 lastmod: 2019-11-21 12:15:08 status_changed: 2019-11-20 14:01:59 type: doctoralThesis metadata_visibility: show creators_name: Vorländer, Matthias Kopano title: Structural studies of RNA Polymerase III transcription subjects: ddc-000 subjects: ddc-500 divisions: i-140001 adv_faculty: af-14 keywords: cryo-EM, electron microscopy, single particle analysis, transcription, RNA Polymerase, Pol III, TFIIIB, TFIIIC, Maf1 cterms_swd: Transkription cterms_swd: Strukturbiologie abstract: RNA Polymerase III (Pol III) produces small, non-coding RNAs with fundamental functions in the eukaryotic cell, including translation, splicing and protein sorting. While structures of unbound and transcribing Pol III have been solved and provided valuable mechanistic insights into Pol III transcription, snapshots of molecular interactions that underlie Pol III activation and repression are lacking. In this thesis I address these questions with structural studies of the Sarrachomyces cerevisiae Pol III transcription apparatus. I present high-resolution cryo-EM reconstructions of Pol III bound to its principal transcription initiation factor TFIIIB that were used to build atomic models. The complex was observed in different functional states, including two early intermediates in which the DNA duplex is closed, an open DNA complex, and an initially transcribing complex with RNA in the active site. The structures reveal an extremely tight, multivalent interaction between TFIIIB and promoter DNA, and explain how TFIIIB recruits Pol III. Together, TFIIIB and Pol III subunit C37 activate the intrinsic transcription factor-like function of the Pol III-specific heterotrimer to initiate the melting of double-stranded DNA, in a mechanism similar to that of the Pol II system. I further present a high resolution structure of Pol III bound to the negative regulator Maf1, that explains how Maf1 achieves transcription repression by preventing interaction with TFIIIB. Maf1 occupies a position on Pol III that overlaps with the binding site of promoter DNA and TFIIIB. Furthermore, by mimicking the shape and electrostatic charge of a double-stranded DNA backbone, Maf1 further sequesters a mobile domain of Pol III subunit C34, which seals off the active site cleft and makes it inaccessible to bind DNA. Lastly, I describe a recombinant expression system for the six-subunit, 520 kDa transcription factor TFIIIC and subcomplexes thereof. Negative stain electron microscopy of a complex between the tA module of TFIIIC and TFIIIB provide the first molecular insights into how TFIIIC recruits TFIIIB and positions it upstream of the transcription start site. Biochemical experiments further show that the tA module is displaced after or concomitant with Pol III recruitment, establishing it as an assembly factor rather than a bona fide transcription factor. date: 2019 id_scheme: DOI id_number: 10.11588/heidok.00027386 ppn_swb: 1682361438 own_urn: urn:nbn:de:bsz:16-heidok-273869 date_accepted: 2019-05-15 advisor: HASH(0x55fc36db3208) language: eng bibsort: VORLANDERMSTRUCTURAL2019 full_text_status: public place_of_pub: Heidelberg citation: Vorländer, Matthias Kopano (2019) Structural studies of RNA Polymerase III transcription. [Dissertation] document_url: https://archiv.ub.uni-heidelberg.de/volltextserver/27386/1/Thesis_MV_18032019_final-compressed.pdf