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Unveiling RNA polymerase III transcription initiation with cryo-EM

Related publication: Abascal-Palacios G., Ramsay E. P., Beuron F., Morris E. & Vannini A. Structural basis of RNA polymerase III transcription initiation. Nature 553, 301 (2018). DOI: 1038/nature25441

Publication Keywords: RNA Polymerase III; tRNAs; gene transcription; TFIIIB

RNA polymerase (Pol) III is a major determinant of lifespan in eukaryotes (organisms whose cells have a nucleus enclosed within membranes, unlike bacteria). The level of Pol III transcription is tightly linked to the rate of growth, as it is known to play a role in cancer, and neurodegenerative diseases. A better understanding of the structure of Pol III could lead to new therapies for these diseases. However, Pol III is the most complex nuclear RNA polymerase.

A team of researchers from the Institute of Cancer Research in London used cryogenic electron microscopy (cryo-EM) at the electron Bio- Imaging Centre (eBIC) to obtain high-resolution maps of a core RNA Pol III pre-initiation complex, which is the minimum essential complex capable of faithfully initiating Pol III transcription in vitro.

Their results show that the transcription factor TFIIIB recruits Pol III at its target genes and activates it to enable melting of the DNA - the initial process of gene transcription. This explains why Pol III transcription is so efficient. This work unravels the molecular mechanisms underlying the first stages of Pol III transcription, and the general conserved mechanisms of gene transcription initiation.

: Figure 1: Two orthogonal views of the core Pol III PIC, represented as a molecular surface. The template and non-template strands of the promoter DNA are coloured in blue and cyan, respectively. The
Pol III enzyme is depicted in grey, with the exception of subunit C34 which is depicted in yellow. TFIIIB components, Brf1, TBP and Bdp1 are depicted in green, pink and orange, respectively.

The eukaryotic nuclear genome is transcribed by the multisubunit enzymes RNA Polymerase (Pol) I, II and III, which catalyze DNA-dependent RNA synthesis. Pol III transcribes genes encoding short non-coding essential RNAs, such as the entire pool of transfer RNAs (tRNAs), the 5S ribosomal RNA (rRNA) and the spliceosomal U6 small nuclear RNA (snRNA). Pol III genes are essential in all cells and involved in fundamental processes such as ribosome and protein biogenesis, RNA processing, and protein transport. Pol III transcription is tightly co-regulated with Pol I activity, accounting together for up to 80% of nuclear gene transcription in growing cells1. Pol III activity influences TORC1- dependent lifespan in yeast, flies and worms and, given its conservation, it is likely to exert the same effect in vertebrates². Pol III deregulation has been associated with a series of neurogenerative diseases and with cancer onset³.

Pol III, with its 17 subunits and nearly 700 kDa mass, is the most complex nuclear RNA polymerase. Recruitment of Pol III at its target promoters rely on a specific subset of transcription factors, including TFIIIA, TFIIIB and TFIIIC. TFIIIA and TFIIIC complexes can be regarded as ‘peripheral’ recruiting factors for the core transcription factor TFIIIB. At all Pol III-transcribed genes, TFIIIB ultimately recruits Pol III resulting in the formation of a closed pre-initiation complex (PIC) and subsequently, assists Pol III in melting the DNA and inserting the template strand at the active site, which is the transition to an open PIC1,3. While Pol I and Pol II requires additional transcription factors, Pol III only requires TFIIIB in order to drive specific transcription from a TATA-box containing U6 promoter in vitro and does not require the ATP-dependent helicase activity of TFIIH for the closed- to open-PIC transition in vivo, as in the case of Pol II^1,3.

The core Pol III transcription factor TFIIIB is a trimeric complex which consists of TBP, Bdp1 and Brf1. Bdp1 is required for nucleation of the transcription bubble (initial DNA melting) while the N-terminal domain of Brf1 is required for the full extension of the transcription bubble, which results in template strand loading in the active site.

: Figure 2: Zoom in of the DNA path within the core Pol III PIC. The initial DNA melting is generated in the upstream part of the transcription bubble (dashed line) where the C34 winged-helix domains
(WHDs) are repositioned upon Bdp1 binding. Final expansion of the transcription bubble occurs upon binding of the Brf1 N-terminal domain (N-term Brf1) that clears the template strand loading path.

Cryo-EM analysis of a core Pol III PIC, reconstituted in vitro using endogenous S.cerevisiae Pol III, recombinant TFIIIB and a 70 base-pairs double-stranded DNA scaffold encompassing the yeast U6 snRNA promoter, resulted in reconstructions of Pol III PIC in different states in the 3.4-4.0 Å resolution range. In particular, an atomic model of a core Pol III PIC in its open state, where the DNA has been spontaneously melted and loaded in the active site of the Pol III enzyme could be built (Fig. 1).

The structure reveals how the transcription factor TFIIIB tightly encapsulates the promoter DNA around the TATA box, explaining the unusually high stability of the Pol III PIC (Fig. 2). The initial DNA melting in the upstream region of the transcription bubble is elicited by a concerted allosteric mechanism involving Pol III subunits C37 and C34 and the Bdp1 subunit of the transcription factor TFIIIB. Upon binding, Bdp1 and a mobile region of subunit C37, the initiation/termination loop, rigidly position the winged-helix domains of subunit C34, that are highly mobile in the unbound Pol III enzyme, in a conformation competent for the initial DNA melting (Fig. 2). Concomitantly, the unwound DNA strands are stabilised by binding to conserved pockets on opposite sides of the Pol III cleft. Finally, binding of the N-terminal domain of Brf1 allows the DNA template strand to be loaded in the active site (Fig. 2). The conformation of the active site in the open Pol III PIC is virtually identical to the one of elongating Pol III, suggesting that immediately after recruitment Pol III adopts an elongation-ready conformation. Thus, TFIIIB efficiently performs two functions at once: TFIIIB effectively recruits Pol III at its target genes and, concomitantly, activates Pol III for initiation of gene transcription, resulting in RNA synthesis.

In summary, cryo-EM reconstructions allowed us to rationalise the molecular basis of the observed high transcriptional efficiency of the Pol III transcription apparatus and allowed for a comparison with the Pol I and Pol II PICs. The Pol III PIC is topologically related to the Pol II PIC while the Pol I PIC is more divergent.

We are now focussing on obtaining cryo-EM reconstructions of a full Pol III PIC, including the multi-subunit transcription factor TFIIIC, which binds promoter elements within the coding region. This structure will reveal how Pol III can efficiently transcribe a gene without deleteriously colliding with TFIIIC.

References:

Vannini A. A structural perspective on RNA polymerase I and RNA polymerase III transcription machineries. Biochim. Biophys. Acta - Gene Regul. Mech. 1829, 258–264 (2013). DOI: 10.1016/j.bbagrm.2012.09.009
Filer D. et al. RNA polymerase III limits longevity downstream of TORC1. Nature 552, 263–267 (2017). DOI: 10.1038/nature25007
Arimbasseri A. G. et al. RNA Polymerase III Advances: Structural and tRNA Functional Views. Trends Biochem. Sci. 41, 546–559 (2016). DOI: 10.1016/j.tibs.2016.03.003
Kassavetis G. A. et al. Transcription factor TFIIIB and transcription by RNA polymerase III. Biochem. Soc. Trans. 34, 1082–1087 (2006). DOI: 10.1042/ BST0341082

Funding acknowledgement:

Biotechnology and Biological Sciences Research Council (BBSRC) New Investigator Award (BB/ K014390/1), Cancer Research UK Programme Foundation (CR-UK C47547/ A21536) and Wellcome Trust Investigator Award (200818/Z/16/Z).

Corresponding author:

Dr Alessandro Vannini, The Institute of Cancer Research, [email protected]

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Unveiling RNA polymerase III transcription initiation with cryo-EM