Abstract
RNA polymerase III (Pol III) is responsible for transcribing 5S ribosomal RNA (5S rRNA), tRNAs, and other short non-coding RNAs. Its recruitment to the 5S rRNA promoter requires transcription factors TFIIIA, TFIIIC, and TFIIIB. Here, we use cryoelectron microscopy (cryo-EM) to visualize the S. cerevisiae complex of TFIIIA and TFIIIC bound to the promoter. Gene-specific factor TFIIIA interacts with DNA and acts as an adaptor for TFIIIC-promoter interactions. We also visualize DNA binding of TFIIIB subunits, Brf1 and TBP (TATA-box binding protein), which results in the full-length 5S rRNA gene wrapping around the complex. Our smFRET study reveals that the DNA within the complex undergoes both sharp bending and partial dissociation on a slow timescale, consistent with the model predicted from our cryo-EM results. Our findings provide new insights into the transcription initiation complex assembly on the 5S rRNA promoter and allow us to directly compare Pol III and Pol II transcription adaptations.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 2641-2652.e7 |
| Journal | Molecular cell |
| Volume | 83 |
| Issue number | 15 |
| DOIs | |
| State | Published - Aug 3 2023 |
Funding
We thank past and present lab members for advice, assistance, and comments on the manuscript. We thank Jason Pattie for computer support. We thank Janette Meyers, Rose Marie Haynes, and Harry Scott at the PNCC for data collection support. We thank the staff at the Structural Biology Facility (SBF) of Northwestern University for technical support. Y. He was supported by an Institutional Research Grant from the American Cancer Society ( IRG-15-173-21 ), NIGMS ( R01GM135651 and R01GM144559 ), NCI ( P01CA092584 ), and an H Foundation Core Facility Pilot Project Award . R.V. was supported by NIGMS ( R01GM140272 ) and The Searle Leadership Fund for the Life Sciences at Northwestern University . R.V. was supported by the National Institutes of Health grant R01GM140272 . Y. He and R.V. were supported by Chicago Biomedical Consortium with support from the Searle Funds at The Chicago Community Trust. A.T. and A.R . were supported by NIGMS ( 5T32GM140995 ). A portion of this research was supported by NIH grant U24GM129547 and performed at the PNCC at OHSU and accessed through EMSL (grid.436923.9), a DOE Office of Science User Facility sponsored by the Office of Biological and Environmental Research . This work used resources of the Northwestern University Structural Biology Facility, which is generously supported by the NCI CCSG P30 CA060553 grant awarded to the Robert H. Lurie Comprehensive Cancer Center. We acknowledge the use of the Ametek K3 direct electron detector, which was generously provided by Professor Robert A. Lamb, Ph.D., Sc.D., HHMI investigator.
Keywords
- FRET
- RNA polymerase III
- TFIIIA
- TFIIIB
- TFIIIC
- cryo-EM
- transcription
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology