cryo-EM study of transition of transcription initiation to elongation
EMSL Project ID
51303
Abstract
Eukaryotic gene expression by RNA polymerase II (pol II) requires the orchestration of a large number of factors during each stage of transcription. Proper gene regulation underlies organismal development, environmental response, and can be disrupted in disease. The goal of this proposal to determine the mechanisms of pol II transitions between initiation, elongation, and re-initiation by pol II, which we will accomplish through structural dissection of transcription complexes by high-resolution cryo-EM. Pol II and general transcription factors (GTFs) assemble in a transcription pre-initiation complex (PIC), which recognizes promoter DNA before every round of transcription, and then opens the double-stranded DNA to expose and select a transcription start site. The PIC transitions to the initially-transcribing complex (ITC), which is responsible for synthesizing a 20–60 nucleotide long nascent transcript and then subsequently transitions to productive elongation (Fig. 1). This set of transitions is universal across all eukaryotes and involves recruitment and reorganization of highly conserved and essential factors. Thus, the structure of the ITC and the conformational and compositional changes that underlie transitions to elongation or re-initiation will reveal mechanisms of essential steps in eukaryotic gene expression. Overlaid on these transitions in metazoans are many additional regulatory steps, most of which can be targeted by a variety of transcription factors and many of which have altered function in human disease. Recently, my laboratory has developed a yeast in vitro transcription system with ~90% transcription efficiency, allowing access to highly homogeneous native transcription complexes for structural and biochemical studies. Our system represents an important breakthrough, especially considering that traditional in vitro transcription systems function at efficiencies of a few percent at most. The basic complex includes 31 yeast polypeptides, including pol II and five GTFs (TBP, TFIIB, TFIIE, TFIIF, and TFIIH). We will use our assembled transcription complexes to determine the structural basis of the transition from initiation to elongation and reinitiation (Fig. 1).
Biochemical studies have indicated that initiating pol II complexes undergo functional and compositional changes (i.e. release or rearrangement of GTFs) as initially transcribing pol II transitions to elongation complex through formation of one or two dozen or so of phosphodiester bonds. We will determine the structural basis for these changes by exploiting our quantitatively efficient in vitro reconstituted transcription system. Our system allows us to generate distinct transcription complexes stalled at different template positions through the use of chain-terminating 3’-O-methyl NTP, thus generating pol II complexes with differing lengths of RNA. We will determine the structural basis of how the ITC accommodates the extended transcription bubble, how inter-subunit interactions change, and how the transition to elongation and re-initiation is facilitated by: (i) determining structures of the ITC with different lengths of RNA by cryo-EM (ITC+26, ITC+49); and (ii) validating structural changes using cross-linking and mass spectrometry (XL-MS).
Project Details
Start Date
2020-03-15
End Date
2021-03-17
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members