Structural and Kinetic Insights into Trna Promoter Engagement by Yeast General Transcription Factor TFIIIC

Transcription of tRNA genes by RNA polymerase III requires the general transcription factor IIIC (TFIIIC), which recognizes intragenic A-box and B-box DNA motifs of type II gene promoters. However, the underlying mechanism has remained elusive, in part due to missing structural information for A-box recognition. In this study, we use single-particle cryo-EM and single-molecule FRET (smFRET) to reveal structural and real-time kinetic insights into how the 520 kDa yeast TFIIIC complex engages A- and B-box DNA motifs in the context of a tRNA gene promoter. Cryo-EM structures of tauA and tauB subcomplexes bound to the A- and B-box were obtained at 3.7 and 2.5 Angstrom resolution, respectively, while cryo-EM single particle mapping determined the specific distance and relative orientation of the tauA and tauB subcomplexes revealing a fully engaged state of TFIIIC. smFRET experiments show that overall recruitment and residence times of TFIIIC on a tRNA gene are primarily governed by B-box recognition, while footprinting experiments suggest a key role of tauA and the A-box in TFIIIB and Pol III recruitment following TFIIIC recognition of type II promoters. ### Competing Interest Statement The authors have declared no competing interest.