Molecular Mechanisms of Transcription Initiation-Structure, Function, and Evolution of TFE/TFIIE-Like Factors and Open Complex Formation

Abstract

Transcription initiation requires that the promoter DNA is melted and the template strand is loaded into the active site of the RNA polymerase (RNAP), forming the open complex (OC). The archaeal initiation factor TFE and its eukaryotic counterpart TFIIE facilitate this process. Recent structural and biophysical studies have revealed the position of TFE/TFIIE within the pre-initiation complex (PIC) and illuminated its role in OC formation. TFE operates via allosteric and direct mechanisms. Firstly, it interacts with the RNAP and induces the opening of the flexible RNAP clamp domain, concomitant with DNA melting and template loading. Secondly, TFE binds physically to single-stranded DNA in the transcription bubble of the OC and increases its stability. The identification of the β-subunit of archaeal TFE enabled us to reconstruct the evolutionary history of TFE/TFIIE-like factors, which is characterised by winged helix (WH) domain expansion in eukaryotes and loss of metal centres including iron-sulfur clusters and Zinc ribbons. OC formation is an important target for the regulation of transcription in all domains of life. We propose that TFE and the bacterial general transcription factor CarD, although structurally and evolutionary unrelated, show interesting parallels in their mechanism to enhance OC formation. We argue that OC formation is used as a way to regulate transcription in all domains of life, and these regulatory mechanisms coevolved with the basal transcription machinery.

Keywords: RNA polymerase; TFIIE/TFE; archaea; evolution; transcription.

Figure 1. Evolution of the transcription initiation machinery after LUCA.

The universally conserved core RNAP and the transcription elongation factor Spt5 are the only components of the transcription machinery that predate the last universal common ancestor (LUCA) of bacteria, archaea, and eukaryotes. The general transcription factors required for transcription initiation emerged later independently in bacteria and archaea, the two primary domains of life. The emergence of eukaryotes from their “archaeal parent” led to the evolution of additional general transcription factors and those belonging to the RNAPII system are listed.

Figure 2. Transcription initiation in archaea is a recruitment cascade.

Sequential or concomitant binding of TBP and TFB to the TATA-box and BRE nucleates the formation of the ternary complex. RNAP is recruited to this platform to form the preinitiation complex (PIC) in the closed form (closed complex, CC). The third factor TFE binds to the CC and assists conformational changes that facilitate DNA melting resulting in the open complex (OC).

Figure 3. The archaeal core promoter structure.

Alignment of the DNA sequences upstream of TSSs identified individual promoter elements including BRE, the TATA box, the initially melted region (IMR) and the initiator (Inr) surrounding the TSS (+1). The strong Inr signal in Sso is due to the fact that the ATG start codon on most genes coincides with the TSS. Alignment of TSS identified by whole transcriptome sequencing from S. solfataricus [42] and T. kodakarensis [39]. The inserts show the TATA box motifs identified by the program MEME (http://meme-suite.org) in the same dataset. Alignment was performed using WebLogo3 (http://weblogo.threeplusone.com) adjusting to the background GC content for each organism.

Figure 4. Architecture of the complete archaeal open complex.

The OC model encompasses the three archaeal general transcription factors TBP (green), TFB (blue) and TFE (magenta), the RNAP (grey) and TS (dark blue) and NTS (cyan). The overall topology of the archaeal OC is very similar to the human OC structure determined by electron microscopy [14]. The relative orientation of the TFEα eWH domain is somewhat uncertain. This model is based on distance constraints derived from smFRET measurements between fluorescent dye pairs introduced at strategic locations in components of the OC [38]. Interprobe distances were calculated from smFRET measurements and processed using the NPS system [86]. Structural parents to the model included the archaeal Sso RNAP (pdb: 2WAQ), ternary complex DNA-TBP-TFB_core from Pyrococcus woesei (pdb: 1D3U), Sso TFEα eWH domain (pdb: 1Q1H), and yeast RNAPII-TFIIB (pdb: 4BBR) and human TFIIEα ZR domain (pdb: 1VD4).

Figure 5. Conformation of the RNAP clamp in archaea.

(A) Structural alignment of the crenarchaeal (S. solfataricus, pdb: 2PMZ) and euryarchaeal RNAP (T. kodakarensis, pdb: 4QIW) that adopt a closed or open clamp, respectively. (B) Single-molecule FRET measurements on immobilised initiation complexes assembled in the presence of a donor-acceptor-labelled RNAP inform about the conformation of the archaeal RNAP clamp. (C) FRET serves as molecular ruler with high sensitivity in the nanometer-range providing information about the width of the DNA cleft and the conformation of the clamp. (D) Opening and closing of the archaeal RNAP clamp during open formation as revealed by smFRET (colour coding as in A and B; non-template strand in cyan, template strand in blue) [56]. TFE binding and OC formation stimulate opening of the RNAP clamp.

Figure 6. Diversity of TFIIE-like factors in archaea and eukaryotes.

(A) The domain composition of dimeric Sulfolobus TFE combines features of TFIIE and RNAPIII subunits RPC62/39. The additional eWH and WH domains resulting from duplications are depicted in light blue and green, respectively. (B) The gene loss and loss of the [4Fe-4S] cluster and Zn-ribbon domains in TFIIE-related factors is depicted on an updated archaeal phylogeny placing eukaryotes within the archaeal domain [5]. Different archaeal taxonomic groups belonging to the euryarchaeota or the ‘TACK’ superphylum [2] as well as the three classes of eukaryotic RNAP systems are included. In order to depict variation within the eukaryotic domain, S. cerevisiae (y) and human (h) counterparts were included separately. The prediction of conservation, or loss, of metal centres is based on the presence of the conserved cysteine residues required for coordination of Zn ions and [4Fe-4S] clusters.

Figure 7. OC formation as a mean to regulate transcription.

Examples of factors and molecules regulating OC formation from different organisms are shown alongside the proposed mechanism of activation or repression (see text for discussion).

Figure 8. TFE-like factors and CarD both activate transcription by stabilising the OC.

(A) Model of the Methanocaldococcus OC with TFEα shown in a magenta semitransparent surface representation. The TFEα eWH domain is perched on the tip of the RNAP clamp coiled coil (orange) in close contact with the NTS of the promoter. (B) Structure of the bacterial OC from Thermus with CarD shown in magenta semitransparent surface representation (pdb: 4XLR) [76]. A conserved tryptophan residue (Trp86) wedges into the minor groove of the upstream DNA thereby stabilising the transcription bubble (see close up).