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Structural studies of RNA Polymerase III transcription

Vorländer, Matthias Kopano

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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.

Item Type: Dissertation
Supervisor: Müller, Dr. Christoph W.
Place of Publication: Heidelberg
Date of thesis defense: 15 May 2019
Date Deposited: 20 Nov 2019 14:01
Date: 2019
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Subjects: 000 Generalities, Science
500 Natural sciences and mathematics
Controlled Keywords: Transkription <Genetik>, Strukturbiologie
Uncontrolled Keywords: cryo-EM, electron microscopy, single particle analysis, transcription, RNA Polymerase, Pol III, TFIIIB, TFIIIC, Maf1
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