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Abstract

The 17-subunit RNA polymerase III (Pol III) complex transcribes short, structured, and essential RNAs like tRNAs. Mis-regulated Pol III is associated with various genetic diseases including cancer and rare developmental diseases. Pol III is also highly specialized to produce short and abundant transcripts, and a key specialization is transcription termination. Pol III terminates on poly-thymidine (dT) sequences on the non-template (NT) strand of the DNA in the absence of any co-factors, which allows fast recycling of Pol III to start a new transcription cycle. Despite recent advances in the structural characterization of yeast Pol III, little is known about the structure of human Pol III, which hinders studying the role of Pol III in human and health and disease. Furthermore, the molecular mechanism of Pol III transcription termination is not known. Here, I present molecular insights into the structure and function of human and yeast Pol III, which I obtained via cryo-electron microscopy (cryo-EM), to address these questions.

I first report the cryo-EM structures of native human Pol III reaching a resolution up to 2.8 Å. The structures shed light on multiple structural features that are different from yeast Pol III. The human RNA-cleaving subunit RPC10 adopts different conformations and can insert into the Pol III active site. A C-terminal extension of subunit RPC5 that is absent in yeast Pol III binds the Pol III core and might function in transcription initiation of metazoan Pol III. An iron-sulphur cluster bound to the human Pol III heterotrimer subcomplex links the Pol III heterotrimer to the Pol III core. The subunit RPC7$\upalpha$, which is associated to cancer, docks onto the Pol III clamp domain and might, thereby, interfere with the inhibition by the repressor MAF1. I also demonstrate that the human Pol III complex can be produced recombinantly, which will enable an in-depth functional analysis of structure and regulation of human Pol III.

Secondly, I report the cryo-EM structure of the yeast Pol III pre-termination complex (PTC) at 2.8 Å resolution. The structure reveals how Pol III recognizes the poly-dT termination signal. The formation of multiple hydrogen-bonds between a set of highly conserved residues in subunit C128 and the termination signal explains how Pol III pauses prior to termination, which I confirm via structure-function studies. NT-strand recognition induces a contraction of the Pol III core, which is reversed in the absence of the C53-C37 heterodimer, explaining its key function in transcription termination. Finally, the structure of the Pol III PTC in the presence of nucleotides shows how Pol III gets trapped in a non-productive state once it reaches the termination signal. Based on these findings, I propose a structure-based mechanism of Pol III transcription termination.