The archaeal transcription apparatus

Today, archaea are known in an astounding diversity of habitats, including the furthest extremes of temperature, pressure, salinity and acidity showing their great ability to adapt to very different living spaces. Their transcription and gene regulatory systems, being hybrids of bacterial and eukaryotic components [17;18; 42;43] (see also section1.1.4), play a very significant role in our efforts to understand the universal aspects of transcriptional mechanisms.

Analyses of the archaeal transcription initiation machinery have revealed striking parallels with the eukaryal RNA polymerase II (RNAP II) transcription apparatus (see section1.1.6.2).

For beeing able to compare the similarities between the archaeal and eukaryal transcription mechanisms a brief summary of the principal steps in transcription initiation by the RNAP II machinery will be given in the following section.

1.1.6.1 The eukaryal RNAP II machinery

The initiation process in eukaryotes starts with the recognition of a sequence element (the TATA box) located∼30bp upstream of the initiation site, by the highly conserved TATA-box-binding

1 Introduction to archaeal transcription and its regulators

protein (TBP), a component of a large multisubunit complex called TFIID. Other subunits of TFIID may additionally contact other promoter elements. A second factor, called TFIIA, can stabilize the binding of the TBP/TFIID complex. After binding, TBP recruits a further factor, TFIIB, serving as a bridge between TBP and the incoming RNA polymerase II (RNAP II). But the main contact between TFIIB and the polymerase appears to be mediated by a factor bound to the polymerase, TFIIF. The complex of TFIID, TFIIB, TFIIF and RNAP II binds tightly to the promoter. For efficient initiation two further factors are required: TFIIE and TFIIH. They facilitate the localized melting of DNA at the start site thus forming the open complex. This pro-cess is ATP driven [44]. They also assist the RNAP II in leaving the promoter thereby abet it in turning from an initiation polymerase to an elongation-competent polymerase. For an overview see figure1.2.

1.1.6.2 The archaeal RNAP machinery

The analysis of the purified archaeal RNA polymerase (RNAP) fromSulfolobus acidocaldarius led to the assumption that the archaeal transcription initiation machinery may be more closely related to that of the eukarya than to that of the bacteria because it could be shown thatS. acido-caldariusRNAP has at least ten subunits [18;45], in contrast to the four-subunit bacterial core enzyme.

Subsequent gene identification for the majority of the subunits of the RNAP of S. acido-caldariusand determination of their sequences showed that the individual subunits are highly conserved in most cases and that the subunit composition is similar to that of eukaryal poly-merases. For instance, the small subunits E, H, K, L and N of the S. acidocaldarius RNAP possess clear homologues in the eukaryal, but not the bacterial, enzymes [46].

Archaeal promoter elements consist of three parts. The typical archaeal promoter contains a TATA-like element (TATA box), ∼25-30bp upstream of the site of transcription initiation [47–49]. The second part of the archaeal promoter element is the BRE (transcription factor B recognition element). It is located immediately upstream of the TATA box and is important for promoter strength as well as the orientation of the transcription initiation complex [50–52].

The presence of these elements combined with the eukaryal-like composition of the poly-merase led to search of a factor homologous to eukaryal TBP. Subsequently, TBP homologues have been identified from a wide range of archaea. Both archaeal and eukaryotic TBP molecules consist of two repeats of about 90 amino acids and adopt a symmetrical saddle-shaped form

1.1 Archaea

Figure 1.2:A: Eukaryal RNAP II machinery. The transcription initiation site is depicted by an arrow. The TFIID complex (blue) binds to the TATA box via its TATA box binding protein component TBP (light blue). TFIIA (pink) stabilizes the protein-DNA interaction. Subsequently TFIIB (green) binds to the DNA-bound TBP and recruits RNAP via interaction with TFIIF (purple). Binding of TFIIE (black) and TFIIH (mauve) to the RNAP leads to promoter melting and clearance. B: Model of the archaeal RNAP machinery. TBP (blue) binds to the TATA box, TFB (green) binds to BRE (not shown) and the RNAP is shown in orange (adapted from Bell and Jackson [45]).

1 Introduction to archaeal transcription and its regulators

Figure 1.3:Picture showing the complex between a DNA fragment displaying BRE (red) and TATA box (ma-genta) and bound TBP (lightblue) and the C-terminal part of TFP (yellow with the helix-turn-helix motif (see section1.3) colored orange. PDB code 1D3U.)

[53; 54]. The similarity of the first to the second repeat is much higher in archaeal TBPs (36-53% of identical amino acids) than in eukaryotic TBPs (22-26%) [55]. Archaeal TBPs are acidic proteins with isoelectric points (IPs) within the range of 3.9-6.1, while the eukaryotic counter-parts are basic, with IPs ranging between 9.8 and 10.7 [56].

In addition to TBP, archaea possess a second general transcription factor, called TFB (Tran-scription factor B). The identification of a partial open reading frame of 152 amino acids with homology to the eukaryotic TFIIB in Pyrococcus woesei was the first indicaton that archaeal transcription factors are of the eukaryotic type [57]. Sequence analysis of the complete gene showed about 30% identity to eukaryotic tfIIbgenes [58]. In addition, this sequence exhibits distinct structural motifs characteristic of eukaryotic TFIIB such as an imperfect amino acid repeat or a zinc ribbon at the N-terminus. InSulfolobus as well as in Methanococcusit could be demonstrated that the archaeal TFIIB homologue (now called TFB) is essential to direct initiation of archaeal transcription [52; 59]. TFB binds to BRE and the N-terminal region is required for RNA polymerase recruitment [60;61]. It could be shown that it is possible to fully reconstitute transcription in the archaeal system using just TBP, TFB and highly purified RNAP [52;62].

Archaeal genome sequencing projects have revealed that most likely all Archaea have a

tran-1.1 Archaea

scription factor resembling TFIIE of the eukaryal system, called TFE. The eukaryotic protein is a heterotetramer composed of two 57kDa and two 34kDa subunits [63;64]. Purified TFIIE has been found to possess no enzymatic activity, it stabilizes the preinitiation complex by binding to the complex as well as to the DNA, and it is involved in the transition from initiation to elon-gation [65;66]. Mutational analysis revealed that the N-terminal half of TFIIEαis sufficient for both basal and activated transcription [67]. Similar results were found in yeast usingin vivo ge-netic experiments [68]. Interestingly, this part of theαsubunit is still conserved in archaea. The crystal structure of the N-terminal domain of TFE fromSulfolobus solfataricusshowed that this domain adopts an extended winged fold with unusual features that are consistent with a role of this domain as an adapter between RNA polymerase and general transcription factors [69]. In vitrotranscription experiments indicate that TFE is not absolutely required for transcription in a reconstituted archaealin vitrosystem; it nonetheless plays a stimulatory role on some promoters and under certain conditions [69].

So the core of the archaeal transcription apparatus consists of an eukaryotic RNAP II-like transcriptase, and the two initiation factors TBP and TFB (see figure1.2).

Archaea initiate transcription by the binding of TBP to the TATA box [59; 70]. Structural analysis of TBP-TATA box co-crystals revealed the mechanism of binding in more detail [71;

72]. Like its eukaryotic counterparts, PyrococcusTBP binds to the minor groove of the DNA and imposes a similar severe distortion on the DNA [72]. Several studies with eukaryotic TBPs suggest that TBP can bind in both orientations with only minimal preference toward the cor-rect orientation [73; 74]. Due to the greater symmetry of archaeal TBPs it is most likely that archaeal TBPs cannot select the right orientation of binding to the TATA box. The polarity of the initiation complex is fixed in the next step by the binding of TFB [50]. It recognizes the distorted DNA-TBP complex and interacts with BRE and the TFIIB-related C-terminal domain of TFB [75;76]. Bound TFB enables the recruitment of the RNA polymerase and the formation of an initiation complex.

Notably, archaeal RNAP subunits A lack the C-terminal repeat extension of eukaryal RNAPII subunit 1 that serves as the assembly platform for complexes that mediate transcriptional activa-tion, chromatin modificaactiva-tion, transcriptional elongation and termination as well as co-transcrip-tional RNA processing. The opening of archaeal promoters by their cognate core transcription machinery is, in contrast to the eukaryal one, not ATP hydrolysis-driven [77].

The fact that extrinsic initiation factors, TBP and TFB, are used in archaeal transcription

1 Introduction to archaeal transcription and its regulators

generates the potential for stably marking a promoter for activity through TBP attachment to the TATA box. In contrast, eukaryotic transcription can utilize cis-acting mechanisms for gen-erating persistent active states of promoters [78; 79]. This led to the statement that there is a

"fundamentally different logic of eukaryotic and bacterial transcription" [80], because in order to establish stable states of actual or potential transcriptional activity eukaryotes have to ad-ditionally use chromatin modification-driven mechanisms which are not part of the bacterial machinery (and almost certainly not part of the archaeal machinery) [77].

Since in bacteriaσleaves the promoter after each round of transcription, constitutive promot-ers are unmarked. So principally successive rounds of transcription at these promotpromot-ers could be uncorrelated. Nevertheless there are feedback mechanisms correlating successive rounds of transcription even in bacteria shown by the fact that active transcription entrains continueing transcription [81].

As to archaea, the behaviour of TBP determines the way archaeal activators and repressors of transcription can exert their regulating effects. If TBP remains stably bound to the promoter repressors will have to block TFB or RNAP entry to the promoter and the repressors themselves must not be excluded by prebound TBP, in order to generate a fast response to signals of envi-ronmental changes. Conversely, if repressors are to be barred by pre-bound TBP, then operators that overlap the transcriptional start site or BRE will lead to faster response to external signals than operators overlapping the TATA box.