title: Structural studies of RNA Polymerase III transcription
creator: Vorländer, Matthias Kopano
subject: ddc-000
subject: 000 Generalities, Science
subject: ddc-500
subject: 500 Natural sciences and mathematics
description: 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
type: Dissertation
type: info:eu-repo/semantics/doctoralThesis
type: NonPeerReviewed
format: application/pdf
identifier: https://archiv.ub.uni-heidelberg.de/volltextserverhttps://archiv.ub.uni-heidelberg.de/volltextserver/27386/1/Thesis_MV_18032019_final-compressed.pdf
identifier: DOI:10.11588/heidok.00027386
identifier: urn:nbn:de:bsz:16-heidok-273869
identifier:   Vorländer, Matthias Kopano  (2019) Structural studies of RNA Polymerase III transcription.  [Dissertation]     
relation: https://archiv.ub.uni-heidelberg.de/volltextserver/27386/
rights: info:eu-repo/semantics/openAccess
rights: http://archiv.ub.uni-heidelberg.de/volltextserver/help/license_urhg.html
language: eng