Structural Studies of RNA Polymerase II and RNA Polymerase III Transcription Initiation


Transcription plays a pivotal role in the transfer of genetic information within living organisms. It serves as the initial step in gene expression, allowing cells to convert the instructions encoded in their DNA into functional molecules. Eukaryotic transcription initiation is a complex and dynamic process that requires joint efforts from many proteins. In this work I explored the intricate mechanisms underlying this process, employing state-of-the-art cryo-electron microscopy (cryo-EM) and single-molecule techniques to visualize the interplay of molecules involved in RNA Polymerase (Pol) II and III transcription initiation.Pol II synthesizes messenger RNA (mRNA) molecules, ultimately enabling the expression of protein-coding genes. The Pol II coactivator Mediator is recruited by transcription factors, facilitating the assembly of the preinitiation complex (PIC) and stimulating phosphorylation of the Pol II C-terminal domain (CTD) by the TFIIH cyclin-activated kinase (CAK) module. I present the cryo-EM structure of the human Mediator-bound PIC. The precise orientation of the CAK within the Mediator-PIC is revealed, with clear density for the Pol II CTD in the active site. A second CTD binding site between Mediator Head and Middle demonstrates how Mediator positions the rest of the CTD for phosphorylation by CDK7. Many regions of Mediator that interact with transcription factors are flexibly tethered, facilitating its assembly. The structure also provides key insights into the conformational landscape of Mediator relative to the PIC. Pol III is responsible for transcribing 5S rRNA, tRNAs, and other short non-coding RNAs. Its recruitment to the 5S rRNA promoter requires transcription factors TFIIIA, TFIIIC, and TFIIIB. I use cryo-EM to visualize the S. cerevisiae complex of TFIIIA and TFIIIC bound to the promoter. Partial TFIIIB binding further stabilizes the DNA, resulting in the full-length 5S rRNA gene wrapping around the complex. Single-molecule FRET (smFRET) experiments reveal that the DNA undergoes both sharp bending and partial dissociation on a slow timescale, consistent with the model predicted from our cryo-EM results. These findings provide new insights into the mechanism of how the PIC assembles on the 5S rRNA promoter, a crucial step in Pol III transcription regulation.

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