RNA Polymerase III Transcription
The research focus of our group is to understand the mechanism of RNA polymerase III (Pol III) transcription. Pol III transcribes tRNAs, 5S ribosomal RNA, and certain small nuclear RNAs. Transcription factors TFIIIA, TFIIIB, and TFIIIC recruit Pol III to assemble the initiation complex at the gene promoter, and subsequently this complex undergoes conformational change to position the DNA template within the Pol III active center for initiating RNA synthesis. The initiation complex relies on specific inter-molecular interactions to organize its functional structural assembly; however, most of these protein/protein and protein/DNA interactions remain to be characterized. In order to provide a detailed protein network for the Pol III initiation complex, we apply site-specific photocrosslinking and hydroxyl-radical probing analyses to map the binding targets for individual amino acids in transcription factors and subunits of Pol III. The biochemical probing is conducted within functional Pol III initiation complex assembled in vitro using proteins from the model organism Saccharomyces cerevisiae (yeast). Our ongoing site-directed biochemical projects are briefly described as follows:
(A) TFIIIB subunits: TBP, Brf1, and Bdp1
A series of TBP single cysteine variants were generated to conjugate with the hydroxyl radical reagent FeBABE at cysteine positions on the convex surface of TBP. These FeBABE-TBP mutants allow us to map the TBP binding sites within TFIIIB subunits Brf1 and Bdp1 to explain how this conserved domain functions in establishing the Pol III-specific protein-DNA structure upstream of the transcription start site. We are also using the non-sense suppressing method to incorporate the non-natural amino acid photocrosslinker p-Benzoyl-L-Phenylalanine (BPA) in positions of Brf1 and Bdp1. By utilizing these BPA-Brf1 and -Bdp1 mutants in initiation complex formation and photocrosslinking, we can provide a more complete protein interactions for Brf1 and Bdp1 to understand how these proteins involves in connecting the transcription factors TFIIIB and TFIIIC for Pol III recruitment.
(B) Pol III subunits: Rpc1, Rpc2, Rpc53, and Rpc37
Rpc1 and Rpc2 form the active site cleft of Pol III and are likely in close contacts with subunits of general transcription factors to position the promoter DNA. The photcrosslinker BPA is incorporated into Rpc1 and Rpc2 to identify the general initiation factors and Pol III subunits interacting with the Pol III active site cleft. The same approach is also applied to study the Pol III-specific subunits Rpc53 and Rpc37, both predicted to locate adjacent to the active site cleft. The protein interaction network derived from this study could explain how Pol III utilizes a specific set of polypeptides to regulate RNA synthesis in its active site cleft.