Preview

PDF, English Download (8MB) | Terms of use

Abstract

RNA polymerase III synthesizes various small non-coding RNAs, including 5S rRNA, tRNA, VAI, and U6 small nuclear RNA, which require different promoter types and auxiliary transcription factors. Transcription of tRNA genes occurs from type 2 promoters and is mediated by transcription factors (TF) IIIB and TFIIIC. Recognition of intragenic A- and B-box motifs in tRNA genes by TFIIIC subcomplexes τA and τB is the first critical step of tRNA synthesis but is mechanistically poorly understood. Despite existing structural information on some individual TFIIIC subunits and τA subcomplex, comprehensive structural data on the complete TFIIIC complex and its interaction with promoter regions was lacking. My work bridges this gap by presenting, for the first time, high-resolution cryo-EM structures of both human and yeast TFIIIC complexes. In this study, I elucidated the structures of human TFIIIC with and without DNA, achieving high-resolution maps ranging from 3.2 to 3.5 Å. These findings unveil the subunit composition of τA and τB subcomplexes in high resolution. A significant revelation was the identification of TFIIIC220's critical role in both subcomplexes and the localization of the flexible linker, which facilitates TFIIIC's binding to its distinct A- and B-box promoters. This was determined through an in-depth analysis of cryo-EM data, including measurements of the distances between τA subcomplex and τB-dimer particles. Subsequently, I proposed a mechanistic model of TFIIIC's interaction with tRNA genes, suggesting that τB, upon recognizing the B-box, anchors the complex to the DNA, thereby enabling the τA subcomplex to engage its promoter through a fly-casting mechanism. Furthermore, I solved the structures of yeast TFIIIC fully bound to DNA at resolutions of 2.67 Å (τB –DNA complex) and 3.73 Å (τA-DNA subcomplex). These structures not only corroborate a similar promoter recognition mechanism as observed in humans but also provide novel insights. The τB-DNA complex sheds light on promoter recognition, and the function of the flexible linker, akin to TFIIIC220 in humans, is observed in τ138. The τA-DNA complex, for the first time, provides insights into this highly dynamic interaction. This interaction was further validated through single-molecule fluorescent microscopy experiments. Overall, my work provides unprecedented structural insights into the TFIIIC complex, significantly advancing our understanding of its interaction with DNA and its role in the transcription of tRNA genes.