Structural visualization of transcription initiation in action

Transcription initiation is a complex process, and its mechanism is incompletely understood. We determined the structures of de novo transcribing complexes TC2 to TC17 with RNA polymerase II halted on G-less promoters when nascent RNAs reach 2 to 17 nucleotides in length, respectively. Connecting these structures generated a movie and a working model. As initially synthesized RNA grows, general transcription factors (GTFs) remain bound to the promoter and the transcription bubble expands. Nucleoside triphosphate (NTP)–driven RNA-DNA translocation and template-strand accumulation in a nearly sealed channel may promote the transition from initially transcribing complexes (ITCs) (TC2 to TC9) to early elongation complexes (EECs) (TC10 to TC17). Our study shows dynamic processes of transcription initiation and reveals why ITCs require GTFs and bubble expansion for initial RNA synthesis, whereas EECs need GTF dissociation from the promoter and bubble collapse for promoter escape. Editor’s summary In eukaryotic cells, general transcription factors (GTFs) support RNA polymerase (Pol) II to assemble a preinitiation complex on a core promoter, open double-stranded DNA, and start RNA synthesis. Chen et al. determined the structures of 16 de novo transcribing complexes, TC2 to TC17, containing nascent RNAs of two to 17 nucleotides, respectively. Connecting these structures revealed an abrupt transition from initially transcribing complexes (in TC2 to TC9, GTFs remained bound to the promoter and Pol II) to early elongation complexes (in TC10 to TC17, GTFs dissociated from the promoter). This transition was accompanied by remarkable conformational and compositional changes, including transcription bubble collapse and GTF dissociation, which allowed Pol II to escape the promoter for product RNA synthesis. —Di Jiang Connecting the structures of 16 de novo transcribing complexes provides molecular visualization of transcription initiation. INTRODUCTION To achieve sophisticated transcription regulation, eukaryotic RNA polymerase II (Pol II) does not open the core promoter by itself and requires general transcription factors (GTFs) (~38 polypeptides in total) to assemble a preinitiation complex (PIC). The PIC then sequentially transforms into an open-promoter PIC complex (OC), an initially transcribing complex (ITC), and an early elongation complex (EEC). Previous studies reported that notable functional and compositional changes occurred during these processes. However, the underlying molecular mechanisms remain largely elusive. RATIONALE We reconstituted the de novo transcribing complexes (TC2 to TC17) with Pol II halted on G-less promoters when nascent RNAs reach 2 to 17 nucleotides (nt) in length, respectively. We determined the cryo–electron microscopy (cryo-EM) structures and recapitulated the dynamic processes of transcription initiation by connecting structures of the PIC (Protein Data Bank ID 7EGB) and 16 TC complexes. RESULTS Structural analyses reveal marked complex reorganizations. Starting from the PIC, the GTF TFIIH couples the energy of adenosine triphosphate (ATP) hydrolysis to translocate the downstream promoter to Pol II, resulting in an OC that contains a transcription bubble of ~13 nt with the transcription start site (TSS) positioned at the active site. The OC then transforms into the ITC (TC2 to TC9), during which the upstream promoter and GTFs remain bound to Pol II, and thus the transcription bubble expands from 14 to 21 nt within the active center. The melted template strand guides the synthesis of nascent RNA, which increases its association with Pol II as it grows from 2 to 9 nt, which is correlated with a transition from abortive transcription to escape commitment. The differences between TC9 and TC10 reveal a sharp ITC-EEC transition and suggest that nucleoside triphosphate (NTP)–driven RNA-DNA translocation and template-strand accumulation in a nearly sealed channel may balloon out the channel, resulting in GTF dissociation, bubble collapse, and promoter escape. The structures of TC10 to TC17 reveal EEC conformation, indicating that Pol II escapes the initiation region and proceeds on the promoter, followed by pausing, RNA capping, pause-release, and productive elongation. CONCLUSION GTFs help Pol II open the core promoter through multiple interactions, which also create an obstacle to the escape of Pol II from the promoter and therefore must eventually be disrupted. Our study provides structural visualization of transcription initiation in action for understanding why and how transcription machinery undergoes such notable changes. Schematic model of transcription initiation. Cryo-EM maps of nucleic acids are shown around the perimeter. Schematic models of representative complexes and nucleic acids are shown in the outer and inner rings of the central area, respectively. Functional states of Pol II and the transcription bubble are indicated. Directions of DNA translocation are indicated with arrows. IIB, IID, IIE, IIF, and IIH are transcription factors TFIIB, TFIID, TFIIE, TFIIF, and TFIIH, respectively. AS, active site of Pol II; NT, nontemplate strand; T, template strand; TBP, TATA box binding protein.