Structural and functional characterization of the human and yeast TFIIIC complex

Abstract

TFIIIC is one of the conserved transcription factors required for RNA Polymerase (Pol) III transcription, which transcribes DNA into untranslated RNAs, such as tRNAs. However, TFIIIC's mechanistic role in Pol III regulation is poorly understood, partly due to the absence of high-resolution structures. In particular, it is unclear how TFIIIC accommodates variable spacings between its recognition motifs (A and B box) on the DNA. To better understand TFIIIC's role in Pol III regulation, I biophysically and structurally characterized the human and yeast TFIIIC complexes. I was able to recombinantly express and purify the human and yeast TFIIIC complexes, enabling the study of preinitiation complexes at tRNA genes in eukaryotes. Preliminary structural analysis of the human TFIIIC complex suggested a two-domain organization, as suspected previously. Contrary to the hypothesis that the B box was indispensable for DNA binding, interaction studies suggested that the A box element was sufficient for DNA binding. Studies of the apo yeast TFIIIC complex via native mass spectrometry and SAXS revealed that it is a flexible complex adopting several conformations. This behaviour was confirmed by cryo-EM: multi-body refinements and hierarchical masking suggested extensive flexibility between and within the two domains of TFIIIC. Despite this, it was possible to obtain an overall reconstruction of the apo-complex at 15A, and sub-nanometre resolution reconstructions of parts of the complex. SAXS and cryo-EM studies suggested that after DNA binding, TFIIIC retained its flexibility while extending further than apo TFIIIC complexes. In conclusion, the presented work has demonstrated the extreme flexibility and heterogeneity of TFIIIC. It has revealed a first glimpse of apo and DNA-bound TFIIIC structures, which may facilitate solving near-atomic resolution structures in the future. This improves our understanding of Pol III transcription in eukaryotes generally and could help to understand how this regulation breaks down in cancerous cells.