Group of Prof. Dr. M. Thomm
Group member: Sebastian Grünberg

New insights into the process of initiation and elongation in a polII-like transcription system


The ability to reconstitute a pure and highly active RNA polymerase (RNAP) brought us in a position to examine the functions of single subunits of the 11-subunit Pyrococcus furiosus RNAP holo-enzyme. The holo-enzyme is composed of a 9-subunit core-enzyme, comprising the major groove and the catalytic centre, and the E’/F-heterodimer, which is the homologues to the eukaryotic Rpb7/4 subcomplex and only loosely bound to the core-enzyme.Within this project we were able to dissect the function of this subcomplex in transcription initiation.

In vitro transcription assays pointed out that under optimized conditions, the level of RNA synthesis by the core-enzyme equals the transcriptional activity of the reconstituted wild type RNAP, while lower incubation temperatures abolished the core-activity. This effect could be compensated by the addition of subunit E’. Permanganate (KMnO4)-footprinting assays revealed that the E’-induced stimulation of the transcription is based on an extension of the transcription bubble’s downstream border. Together with these results, analyses of the polII crystal structure suggest a role for E’ in closing the mobile clamp of the RNAP (Fig. 1), accompanied by the melting of the template DNA around the active centre of the RNAP and downstream of that region, respectively.

E'/F induced closure of the RNAP clamp

Fig. 1: A, Structure of the RNAP core-enzyme (grey) with attached Rpb7 (blue) /4 (dark red) heterodimer. The coding strand is highlighted in light blue; the non-coding strand is identified in pink. The clamp is bordered in black. B, Schematic view of A. The colouring of the Rpb7/4-subcomplex resembles the colouring in A, the active centre of the RNAP is highlighted by a red dot. The clamp is illustrated in both, the E’-induced closed conformation (continuous line) and the opened conformation (dashed line), respectively.


Analyses of the archaeal transcription factor E (TFE), the homologue of the N-terminal domain of the general polII transcription factor E (TFIIE) alpha-subunit, revealed a dual role of this factor in initiation reactions.

First, TFE stabilizes preinitiation complexes (PICs) formed by the core-enzyme, as assayed in heparin-competition experiments. Second, TFE dramatically stimulates the E’-dependent activity of the core-enzyme. KMnO4-footprinting studies showed that this stimulation is based on the stabilization of the transcription bubble by TFE as well as an active TFE-induced opening of the DNA at the upstream border of the bubble. In cooperation with Mike Bartlett’s group (Portland State University, Portland, Oregon, USA) we were able to document direct binding of TFE to the surface-exposed non-coding strand in PICs, resulting in increased stability and further extension of the bubble.Summarizing the results mentioned above, we were able to shed new light on the process of initiation of a polII-like transcriptional machinery (Fig. 2). After core-RNAP recruitment to the promoter platform (A) a minimal open complex is formed (B). Binding of E’/F induces clamp closure, resulting in an extension of the transcription bubble towards the transcription start site (C) and this step is crucial for transcriptional activity. TFE is recruited to the PIC and interacts with the non template strand in the region upstream of the start site, accompanied by an active opening of the DNA at the upstream border of the bubble, resulting in a full open complex (D).

Initiation of transcription

Fig. 2: Schematic view of the different steps during initiation. A, The core-enzyme (blue) is recruited to the promoter platform (TBP, green; TFB, grey). After the reorganization of the binary complex the core-enzyme is able to form a minimal open complex (B). Binding of the E’/F-heterodimer to the core-enzyme induces clamp (red) closure, which is accompanied by an extension of the transcription bubble in direction of the RNAP’s active centre (C). D, TFE interacts with the non-coding strand upstream of the catalytic centre, resulting in a stabilization of the bubble as well as an active opening of the DNA at the upstream border of the bubble.


Additionally, photochemical cross-linking studies uncovered a novel role of TFE, which seems to be unique for this primary member of the TFE-family. While the eukaryotic counterpart of TFE, TFIIE, is released during transition from initiation to elongation, archaeal TFE remains RNAP-bound. As part of the elongating complex, TFE stabilizes the transcription bubble in ternary complex due to direct interactions with the non-coding strand in the region upstream of the active centre (Fig. 3). Like in the eukaryotic polII-system, TFE can not subsequently be recruited to elongating complexes. The recruitment of TFE to the PIC is a prerequisite for the stabilizing effect of TFE on the transcription bubble in ternary complexes.

Elongation complex, stabilized by TFE

Fig. 3: Schematic view of an elongation complex, stabilized by TFE (RNAP, blue; TFE, yellow; non template strand, red; template strand, black). The black bar represents the pausing position and the active centre of the ternary complex, respectively. TFE directly interacts with the DNA of the non-template strand in the region of the open bubble.