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Recent advances in the understanding of archaeal transcription
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Recent advances in the understanding of archaeal transcription Dina Grohmann and Finn Werner RNA polymerases (RNAPs) make repeatedly use of their templates by cycling through initiation, elongation and termination phases of transcription; during each step RNAP is interacting with and regulated by distinct transcription factors. The dynamic interplay between nucleic acid sequences, transcription factors and RNAP affects the activity and distribution of transcription complexes across the genome, and ultimately executes the genetic programme of the organism. This review covers recent discoveries about the mechanisms of archaeal transcription obtained by a combination of in vivo and in vitro approaches, from the molecular to the global level. Address RNAP Laboratory, Institute for Structural and Molecular Biology, Division of Biosciences, University College London, Darwin Building, Gower Street, London WC1E 6BT, United Kingdom Corresponding author: Grohmann, Dina ([email protected])
Current Opinion in Microbiology 2011, 14:328–334 This review comes from a themed issue on Archaea Edited by John Reeve and Christa Schleper Available online 17th May 2011 1369-5274/$ – see front matter # 2011 Elsevier Ltd. All rights reserved. DOI 10.1016/j.mib.2011.04.012
Introduction The last five years have witnessed the birth of archaeal genetics and systems biology and a combination of both with biochemical approaches has added to our understanding of differential gene expression in Archaea in response to environmental changes. Gene expression is controlled by the DNA sequence of the genome, by general and gene-specific transcription factors, by the RNAP and potentially by non-coding (nc) RNAs; Table 1 lists various mechanisms that are discussed in this review.
The RNAP transcription apparatus The archaeal transcription machinery is closely related to the eukaryotic systems in terms of RNAP structure and function, but relies on a minimal set of transcription factors and promoter elements (Figure 1a). Moreover, the molecular mechanisms accompanying the transcription cycle (Figure 1b) are conserved [1]. Archaeal RNAPs consist of 11–12 subunits that are structurally and functionally homologous to eukaryotic RNAPII subunits. The Current Opinion in Microbiology 2011, 14:328–334
core of the enzyme consists of the catalytic Rpo1 and Rpo2 subunits and an assembly platform made of Rpo3, Rpo10, Rpo11 and Rpo12. Rpo1 does not contain the Cterminal domain (CTD) common to its eukaryotic counterpart RBP1 and therefore lacks a target for many regulatory factors. A group of small RNAP subunits, which are not strictly required for basic RNAP function including promoter directed transcription, are incorporated into the enzyme close to the RNA exit channel (Rpo4/7), proximal to the NTP entry pore (Rpo8) and at the tip of the RNAP jaws (Rpo5 and Rpo13). Rpo4 and Rpo7 form the RNAP stalk, which is anchored to the RNAP via Rpo6 (Figure 2a). The stalk has emerged as an important feature of RNAP for all phases of the transcription cycle [2]. During initiation it promotes DNA melting and interacts with TFE [3–6]. During elongation the stalk interacts with the RNA transcript via an S1 motif in Rpo7 and not only facilitates robust processivity but also enhances transcription termination at weak termination signals [7]. The structure of the Rpo4/7 complex suggests that Rpo4 stabilises Rpo7 [8]. Genetic investigations have shown that whereas the archaeal rpo7 and the eukaryotic rpb7 are essential genes, archaeal rpo4 and eukaryotic rpb4 can be deleted with viability retained at moderate temperatures [6,9]. In addition to the RNAP stalk, mutations in the switch 3 motif in the Rpo2 subunit also affect the transcription elongation rate, possibly by promoting strand separation at the upstream edge of the DNA– RNA hybrid [10]. Phosphodiester bond formation and the mechanism of translocation that facilitates the movement of RNAP along the DNA template are facilitated by the active site. The RNAP bridge (Figure 2a, orange) and trigger helices are intimately involved in this process, and a saturation mutagenesis approach has provided exciting evidence that ‘superactive’ RNAP mutants correlate with distinct conformational states of the bridge helix [11,12].
Mechanisms of basal initiation factors Archaeal RNAP has a strict requirement for two basal factors, TBP (TATA binding protein) and TFB (transcription factor B) for transcription initiation [13,14] and highly conserved homologues of these factors, namely TBP and TFIIB, are required for eukaryotic transcription initiation. TBP binds to the TATA element of the promoter and distorts its topology by bending it approximately 908 [15]. The directionality of the promoter is brought forth by binding of TFB to the B-recognition element (BRE) upstream of the TATA element [16–18]. The TATA–TBP–TFB platform recruits RNAP forming the minimal preinitiation complex (PIC). In addition to recruiting RNAP, TFB contributes directly to the mechanism of initiation by donating a highly flexible linker www.sciencedirect.com
Understanding of archaeal transcription Grohmann and Werner 329
Table 1 Sequence elements and factors that regulate transcription in Archaea. The interplay between template DNA and transcript RNA sequence, basal and gene-specific transcription factors and the RNA polymerase governs the regulation of transcription. In addition histones and histone-like proteins and predicted non-coding RNAs including antisense transcripts have the potential to direct transcription in a gene-specific manner. Abbreviations: template strand (TS), non-template strand (NTS), B-recognition element (BRE), initiator element (Inr), tetrasome-localising sequence (TLS). The references are examples and not exhaustive. Nucleic acid element Initiation (promoter)
Elongation (intragenic)
Protein factor
TATA BRE
TBP TFB
TS at start site NTS at start site Inr/TS Pause (DNA) Pause (DNA) RNA non-sequence specific
Mechanism
Stimulation/ inhibition
Refs
+/ +/
[13,14] [13,16,17]
TFB TFE RNAP
TBP recruitment TFB and RNAP recruitment/directionality Initiation/catalysis DNA melting Promoter strength
? + +/
[5,19] [5,21] [24,25]
Spt4/5 TFS Rpo4/7
RNAP processivity Transcript cleavage RNAP processivity
+ + +
[32] [56] [7]
Termination (terminator)
poly-dA:rU signal
Rpo4/7
Enhanced termination
?
[7]
Gene-specific regulation
TATA/BRE
TBP/TFB, alternative variants Activator (e.g. Ptr2) Repressor (e.g. MDR1) Histones –
Promoter selection/internal promoters Ptr2: TBP recruitment MDR1: Promoter occlusion Promoter occlusion Hybridisation to mRNA
+/
[30]
+ ?
[57] [58] [49,50] [24,54]
Activator binding motif Repressor binding motif TLS antisense RNAs
region to complement the active site of RNAP proximal to the template strand (TS) [5,19,20]. A third archaeal initiation factor, TFE (transcription factor E), associates with RNAP and stimulates DNA melting (‘open complex formation’), during which the TS is loaded into the active site [5,21,22]. TFE interacts with the non-template strand (NTS) and thereby stabilises the PIC [21]. A recent computational search has identified a putative homologue of TFE beta (or C34, the RNAPIII homologue), but its function as initiation factor still needs to be validated experimentally [23]. In the open complex RNAP interacts with the TS around the transcription start site, which occasionally is referred to as the initiator element (Inr). The Inr has a weak sequence bias (mainly T(1)A(+1) [24]) and variations in its sequence affect the promoter strength, which suggests sequence-specific interactions between Inr and RNAP and/or TFB [5,25]. Many Archaea encode multiple homologues of the general transcription factors TBP and TFB. Halobacterium NRC-1 encodes six TBPs and seven TFB variants [26]. Theoretically this allows for 42 different TFB–TBP combinations that could facilitate differential regulation of transcription by alternative TFB–TBP combinations. In Halobacterium six out of 13 TBP and TFB genes are essential for growth under standard conditions [27]. ChIPchip results suggest that some of the TFB variants are enriched on a subset of promoters of genes with similar functions [28]. Ideally a combination of genetic and biochemical approaches should be used to dissect the contribution of the individual factors to gene-specific regulation. However, just a few organisms with estabwww.sciencedirect.com
lished in vitro transcription system are also genetically tractable with one excellent exception: Thermococcus kodakarensis. Thermococcus encodes two TFB variants, TFB1 and TFB2, and combined genetic and biochemical approaches have shown that both are active and support transcription from a range of promoters even though the optimal salt concentrations differ for the two variants in vitro [29]. However, the deletion of either TFB did not lead to any phenotypes in vivo, which suggests a high degree of redundancy. A deletion analysis of the three TBP homologues in Methanosarcina acetivorans revealed that just TBP1 is essential for survival and TBP2 and 3 play a role in environmental adaptation [30]. In addition to multiple isoforms of full length TBPs and TFBs, some Archaea also encode truncated versions of these transcription factors. For example, ultraviolet radiation of Sulfolobus solfataricus induces the expression of an unorthodox TFB variant, TFB3, which lacks the C-terminal core domain that is responsible for interactions with TBP and the promoter BRE sequence [31]. TFB3 is thus not able to form ternary DNA–TBP–TFB complexes. However, TFB3 competes with the canonical TFB1 for binding to RNAP, and interacts with the ternary TFB1– TBP–DNA complex. TFB3 stimulates transcription from several promoters including genes that are induced by UV irradiation. This stimulating effect is strictly dependent on TFB1, which suggests that TFB3 acts in concert with TFB1 on the same transcription initiation complexes by a mechanism that is not yet understood. Nevertheless, the cooperation of two TFB variants is a feature unique to the archaeal domain of life. Current Opinion in Microbiology 2011, 14:328–334
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Figure 1
RNAP subunits
(a)
Archaea
Eukaryotes
Rpo1 (A) Rpo2 (B) Rpo3 (D) Rpo11 (L) Rpo6 (K) Rpo5 (H) Rpo8* (G) Rpo10 (N) Rpo12 (P) Rpo4 (F) Rpo7 (E)
RPB1 RPB2 RPB3 RPB11 RPB6 RPB5 RPB8 RPB10 RPB12 RPB4 RPB7 RPB9
(b)
transcription factors
Termination
Initiation RNAP recruitment TBP/TFB
ribosome S10
Transcription cycle Initiation DNA melting TFE
Elongation Spt4/5 TFS
Rpo13* TBP TFB TFEα TFEβ/C34* TFS Spt4 Spt5 NusA
Re-initiation
TBP TFIIB TFIIEα TFIIEβ TFIIS Spt4 Spt5
Pause/backtrack/ arrest/termination? Initiation promoter escape
TFE
Spt4/5 Current Opinion in Microbiology
The archaeal transcription cycle. (a) Archaeal RNAP subunits and transcription factors and their eukaryotic RNAPII counterparts. Note that homologues of Rpo8, Rpo13 and TFEbeta/C34 are not present in all archaeal genomes (highlighted with asterisks). (b) RNAP repeatedly makes use of the same DNA template by cycling through initiation, elongation and termination phases. During initiation TBP and TFB assemble on the promoter and recruit RNAP. TFE stimulates the next stage of initiation during which the DNA is melted and the template strand loaded into the active site. Spt4/5 and TFS associate with the elongation complex and stimulate processivity; in addition Spt4/5 has the potential to couple transcription and translation via interactions between the Spt5 KOW domain and ribosomal protein S10 (NusE).
Post-initiation mechanisms of transcription Following promoter escape RNAP enters the elongation phase of transcription (Figure 1b). Elongation is a discontinuous process that is frequently interrupted by pausing, and transcription elongation factors alleviate these impediments. Spt5, a homologue of bacterial NusG, is a universally conserved elongation factor that usually interacts with Spt4 and, when bound to RNAP, increases transcription processivity [32]. The Spt5 NGN (NusGN-terminal) domain is required for this stimulatory activity and interacts with the RNAP clamp coiled coil [32] to form a bridge across the DNA-binding channel. Using the archaeal proteins it has been possible to obtain structural information of the RNAP–Spt4/5 complex for the first time [33,34]. These studies provide insights into the likely mechanism for the enhanced processivity of the RNAP upon interaction with Spt4/5. The structural investigations imply that Spt5 induces the closure of the clamp domain, which might lead to a further encapsulation of the DNA and thereby preventing the release of the DNA template and the dissociation of the elongation complex. The function of the archaeal Spt5 KOW (Kyprides–Ouzounis–Woese) domain is less clear, but in Bacteria the Current Opinion in Microbiology 2011, 14:328–334
NusG KOW domain recruits factors involved in termination/anti-termination mechanisms including the rho helicase and NusE. In Eukaryotes and Bacteria, multiple RNAPs transcribing the same operon/genein the same direction cooperate, most likely by preventing a retrograde movement of RNAP called back-tracking [35,36]. Similar to Bacteria, many archaeal genes are organised in multi-cistronic operons and mRNAs are translated co-transcriptionally. This gives rise to polarity — expression of a downstream gene is affected by genes upstream in the same operon [37]. In Bacteria the ratio of nucleotide and amino acid addition rates remains constant (between 2.9 and 3.1) over a range of addition and growth rates [38]. This adjusts the transcriptional yield to translational needs. A recent report suggests that NusG (Spt5) physically links elongating RNAPs and ribosomes via interactions between the NusG KOW domain and NusE, which is identical to ribosomal protein S10 [39]. This tethering of RNAP and ribosomes explains not only why ribosomes suppress sequence-dependent pausing of RNAP by ‘pushing’ them forward, but also why conditions that decrease translation rates (e.g. growth conditions, codon usage www.sciencedirect.com
Understanding of archaeal transcription Grohmann and Werner 331
Figure 2
(a)
(b) Ribosome 30S subunit structure
model stalk
mRNA transcript Rpo1 (A)
transcription
Rpo2 (B)
clamp
Rpo3 (D)
NusE(S10)
Rpo11 (L)
Spt4
Rpo6 (K)
Spt5 NGN Spt5 KOW
Rpo4 (F) Rpo5 (H)
duplex DNA
RNAP
Rpo7 (E) Rpo8 (G)
3’
Rpo10 (N) Rpo12 (P)
assembly platform catalytic centre
Rpo13
bridge helix
downstream DNA binding site
catalytic centre Current Opinion in Microbiology
Structure and function of RNAP. (a) Important features of RNAP that are discussed in the text are circled in red (assembly platform), blue (stalk), green (clamp) and firebrick red (active site). The downstream duplex DNA is indicated as grey cylinder. RNAP structure is based on Sulfolobus (pdb 2WAQ), the RNAP subunits are colour-coded according to convention [1]. (b) Working model of the Spt4/5 modus operandi. The universally conserved elongation factor Spt4/5 (NusG in bacteria) is recruited to the RNAP clamp coiled coil and closes the DNA-binding channel over the DNA template (duplex DNA indicated as salmon pink double helix, RNA in red). This interaction may deny the dissociation of the template from the elongation complex and thereby enhance processivity. In Bacteria, and likely in Archaea, the NusG KOW domain (green) interacts with ribosomal protein S10 (blue), which tethers elongating ribosomes (30S subunit shown in firebrick red, not drawn to scale) and RNAP.
and antibiotics) also lower transcription elongation rates [38]. Based on the high evolutionary conservation of the Spt5 and NusG KOW domains and S10 it is likely that this mechanism also operates in Archaea (Figure 2b). Transcription termination is the final and least well understood phase of the transcription cycle. Archaeal RNAP appears to terminate via a mechanism similar to that employed by eukaryotic RNAPIII in which short poly-dA stretches in the template DNA (resulting in rU:dA RNA:DNA hybrids) serve as signals for efficient termination in vivo and in vitro, without any requirements for secondary structures in the transcript (such as hairpins), or exogenous termination factors (e.g. the bacterial rho factor) [7,40,41]. The transcription elongation complex is very stable and any model for transcription termination must provide a convincing rationale for its destabilisation and dissociation. This is likely to involve an opening of the RNAP clamp and requires the dissociation of any RNAP-bound Spt4/5 from the elongation complex. Alternatively, recent data suggest that terminated archaeal RNAPs are ‘recycled’ to the promoter and www.sciencedirect.com
re-initiate transcription like the eukaryotic RNAPIII enzyme [42].
Regulation of transcription by chromatin Different Archaea encode many positively charged but evolutionary unrelated DNA-binding proteins including histones [43], Alba [44], Sac7d [45], CC1 [46] and Cren7 [47], which are thought to compact the archaeal DNA in chromatin-like structures. Archaeal histones are smaller than their eukaryotic homologues, but adopt the typical histone fold that is formed by three alpha-helices separated by two short beta-strand loops. They lack the tails that are extensively post-translational modified in eukaryotic histones [48]. While Eukaryotes compact their nuclear DNA employing exclusively the same four histones Archaea often encode more than one histone variant that readily form homodimers and heterodimers. These can assemble into tetramers in the presence of DNA and compact approximately 80 bp of DNA, as compared to the octameric histones that compact 147 bp in Eukaryotes. Even though strict nucleosome positioning sequences have not been identified in Current Opinion in Microbiology 2011, 14:328–334
332 Archaea
Archaea yet, a SELEX approach has identified DNA sequences that preferentially are bound by archaeal histones (tetrasome-localising sequences, TLSs) [49]. This result makes it plausible that sequence-specific chromatin-assemblies can regulate transcription by allowing/denying access of the transcription machinery to the promoters [49,50]. Archaeal histone binding to a template DNA reduces but does not fully inhibit promoterdependent transcription in vitro, except at non-physiologically high histone:DNA ratios. However, the histonemediated transcriptional repression can be relieved in vitro by at least one activator, designated Ptr2 [50]. Most likely Ptr2 competes with histones for the binding to the promoter DNA and, when bound, allows RNAP and basal transcription factors access to the template. Archaeal RNAPs are able to elongate through histone-covered DNA templates, albeit at reduced rates [51]. In summary, the competition between histones, transcriptional activators and transcription initiation factors as shown in in vitro experiments has the potential to orchestrate gene-specific regulation of transcription also in vivo (Figure 3).
Global approaches to archaeal gene expression The prediction of operon organisation is problematic at best, but the deep sequencing of the S. solfataricus transcriptome proved beyond doubt that protein encoding genes in Archaea are organised both in mono-cistronic (72%) and poly-cistronic (28%) operons [24]. Early archaeal transcriptomic experiments used microarray technology and aimed at the detection of changes in gene expression under different growth conditions and environmental insults (e.g. heat shock, osmotic shock, viral infection, for a recent review [52]). In S. solfataricus the operon encoding the large Rpo1 and Rpo2 subunits is down regulated under heat shock conditions, which serves as a fast global shutdown of transcription in response to severe stress [53]. Archaea encode a plethora of ncRNAs. In Sulfolobus at least 310 ncRNAs have been identified, some of which have the potential to regulate gene expression both at the transcriptional and posttranscriptional level. The majority of ncRNAs identified in transcriptomic studies are antisense transcripts of existing operons (Figure 3). A closer scrutiny revealed that these operons primarily encode genes involved in ion transport and metabolism [24]. Microarray transcriptomics in Halobacterium combined with whole genome occupancy profiling (ChIP-chip) showed that Archaea make use of alternative intragenic promoters in a growth phase-dependent manner [54]. Ten percent of the TBP/ TFB transcription factor binding sites map to the coding regions of multi-cistronic operons, and appear to act as additional transcription start sites (Figure 3). Conditional transcription of poly-cistronic operons has been described for the sdhCDBA operon. Whereas sdhCDB (encoding for Current Opinion in Microbiology 2011, 14:328–334
Figure 3
(a)
Internal promoter in a polycistronic unit A
B
C
D
(b)
Antisense transcription
(c)
Regulation by multiple TFB/TBP combinations TBP1/TFB1
(d)
TBP1/TFB3
TBP4/TFB1
Promoter occlusion by histones
X Ptr2 Ptr2
Histone-like protein Current Opinion in Microbiology
Transcriptional regulation. Archaeal poly-cistronic operons have alternative internal promoters (highlighted with a dotted blue arrow) that are subjected to growth phase-dependent regulation (a). Antisense transcripts are synthesised from promoters on the complementary strand of the ORF; they can potentially anneal with the mRNA by Watson–Crick basepairing and affect its translation and/or stability (b). Combinations of TBP and TFB variants may recruit RNAP to subsets of genes and thereby regulate transcription in a gene-specific fashion in archaeal organisms that encode multiple TBP/TFB variants (c). Histonelike proteins have been shown to occlude promoter elements and repress transcription; transcription activators such as Ptr2 can activate transcription by competing with histones for promoter binding (d).
subunits of the succinate-dehydrogenease (SDH) enzyme complex, the Fe–S, cytochrome b and membrane anchor subunits) is down regulated at high cell densities, the expression of sdhA (encoding for the FAD-binding flavoprotein component) remains unchanged. And recent studies on the pst (phosphate-specific transport) operon in Halobacterium salinarum support the finding that Archaea make use of alternative promoters [55]. Here, transcription starts at two different promoters within the multi-cistronic operon in a phosphate-dependent manner. In summary, the transcription of archaeal poly-cistronic operons is fine-tuned and alternative internal promoters are either induced or repressed depending upon environmental conditions.
Perspective Many of the discoveries described in this review add to our understanding about transcriptional regulation in www.sciencedirect.com
Understanding of archaeal transcription Grohmann and Werner 333
Archaea even though some mechanistic details are not elucidated yet. For example the meaning of multiple copies of the transcription factors TFB and TBP encoded in some archaeal organisms remain unclear despite the advances in archaeal genetics.
11. Tan L, Wiesler S, Trzaska D, Carney HC, Weinzierl ROJ: Bridge helix and trigger loop perturbations generate superactive RNA polymerases. J Biol 2008, 7:40. 12. Weinzierl ROJ: Nanomechanical constraints acting on the catalytic site of cellular RNA polymerases. Biochem Soc Trans 2010, 38:428-432. 13. Werner F, Weinzierl ROJ: A recombinant RNA polymerase II-like enzyme capable of promoter-specific transcription. Mol Cell 2002, 10:635-646.
The challenge for the future will be to validate the wealth of information derived from systems biology approaches and to observe the archaeal RNAP encountering ‘roadblocks’ like histones in vivo. This includes for instance, firstly, a detailed description of the molecular mechanisms that couple transcription and translation in Archaea, secondly, to determine whether or not archaeal genomes are compacted by histones and histone-like proteins in vivo and thirdly to specify the function of the ncRNAs identified in transcriptomic studies.
15. Grohmann D, Klose D, Fielden D, Werner F: FRET (fluorescence resonance energy transfer) sheds light on transcription. Biochem Soc Trans 2011, 39:122-127.
Acknowledgements
17. Bell SD, Kosa PL, Sigler PB, Jackson SP: Orientation of the transcription preinitiation complex in archaea. Proc Natl Acad Sci U S A 1999, 96:13662-13667.
Research at the RNAP Laboratory was funded by project grants from the Wellcome Trust (079351/Z/06/Z) and BBSRC (BB/E008232/1 and BB/ H019332/1) to Finn Werner. We apologise to our colleagues whose work could not be cited due to space constraints.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
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2.
Grohmann D, Werner F: Cycling through transcription with the RNA polymerase F/E (RPB4/7) complex: structure, function and evolution of archaeal RNA polymerase. Res Microbiol 2011, 162:10-18.
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Naji S, Gru¨nberg S, Thomm M: The RPB7 orthologue E0 is required for transcriptional activity of a reconstituted archaeal core enzyme at low temperatures and stimulates open complex formation. J Biol Chem 2007, 282:11047-11057. Ouhammouch M, Werner F, Weinzierl ROJ, Geiduschek EP: A fully recombinant system for activator-dependent archaeal transcription. J Biol Chem 2004, 279:51719-51721.
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Hirata A, Kanai T, Santangelo TJ, Tajiri M, Manabe K, Reeve JN, Imanaka T, Murakami KS: Archaeal RNA polymerase subunits E and F are not required for transcription in vitro, but a Thermococcus kodakarensis mutant lacking subunit F is temperature-sensitive. Mol Microbiol 2008, 70:623-633.
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Hirtreiter A, Grohmann D, Werner F: Molecular mechanisms of RNA polymerase — the F/E (RPB4/7) complex is required for high processivity in vitro. Nucleic Acids Res 2010, 38:585-596.
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14. Qureshi SA, Bell SD, Jackson SP: Factor requirements for transcription in the Archaeon Sulfolobus shibatae. EMBO J 1997, 16:2927-2936.
16. Renfrow MB, Naryshkin N, Lewis LM, Chen H, Ebright RH, Scott RA: Transcription factor B contacts promoter DNA near the transcription start site of the archaeal transcription initiation complex. J Biol Chem 2004, 279:2825-2831.
18. Bartlett MS, Thomm M, Geiduschek EP: Topography of the euryarchaeal transcription initiation complex. J Biol Chem 2004, 279:5894-5903. 19. Kostrewa D, Zeller ME, Armache K, Seizl M, Leike K, Thomm M, Cramer P: RNA polymerase II-TFIIB structure and mechanism of transcription initiation. Nature 2009, 462:323-330. 20. Wiesler SC, Weinzierl ROJ: The linker domain of basal transcription factor TFIIB controls distinct recruitment and transcription stimulation functions. Nucleic Acids Res 2011, 39:464-474. This study made us of the recombinant archaeal transcription system from Methanocaldoccus jannaschii to study the influence of the highly flexible TFB-linker on abortive transcription. Saturation mutagenesis of the linker domain, which is located in close proximity to the catalytic site of the RNAP identified the residues that are crucial to the activity of TFB and allowed the identification of ‘super-stimulatory’ TFB-variants. 21. Gru¨nberg S, Bartlett MS, Naji S, Thomm M: Transcription factor E is a part of transcription elongation complexes. J Biol Chem 2007, 282:35482-35490. 22. Bell SD, Brinkman AB, van der Oost J, Jackson SP: The archaeal TFIIEalpha homologue facilitates transcription initiation by enhancing TATA-box recognition. EMBO Rep 2001, 2:133-138. 23. Blombach F, Makarova KS, Marrero J, Siebers B, Koonin EV, van der Oost J: Identification of an ortholog of the eukaryotic RNA polymerase III subunit RPC34 in Crenarchaeota and Thaumarchaeota suggests specialization of RNA polymerases for coding and non-coding RNAs in Archaea. Biol Direct 2009, 4:39. 24. Wurtzel O, Sapra R, Chen F, Zhu Y, Simmons BA, Sorek R: A single-base resolution map of an archaeal transcriptome. Genome Res 2010, 20:133-141. A deep-sequencing study of the transcriptome from Sulfolobus solfataricus that led to the correction of 162 gene annotations, definition of 80 new ORFs, prediction of 80 noncoding RNAs, identification of antisense transcripts and determination of the operon structures of more than 1000 transcriptional units. 25. Qureshi SA: Role of the Sulfolobus shibatae viral T6 initiator in conferring promoter strength and in influencing transcription start site selection. Can J Microbiol 2006, 52:1136-1140. 26. Baliga NS, Goo YA, Ng WV, Hood L, Daniels CJ, DasSarma S: Is gene expression in Halobacterium NRC-1 regulated by multiple TBP and TFB transcription factors? Mol Microbiol 2000, 36:1184-1185. 27. Coker JA, DasSarma S: Genetic and transcriptomic analysis of transcription factor genes in the model halophilic Archaeon: coordinate action of TbpD and TfbA. BMC Genet 2007, 8:61.
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28. Facciotti MT, Reiss DJ, Pan M, Kaur A, Vuthoori M, Bonneau R, Shannon P, Srivastava A, Donohoe SM, Hood LE et al.: General transcription factor specified global gene regulation in archaea. Proc Natl Acad Sci U S A 2007, 104:4630-4635. 29. Santangelo TJ, Cubonova´ L, James CL, Reeve JN: TFB1 or TFB2 is sufficient for Thermococcus kodakarensis viability and for basal transcription in vitro. J Mol Biol 2007, 367:344-357. 30. Reichlen MJ, Murakami KS, Ferry JG: Functional analysis of the three TATA binding protein homologs in Methanosarcina acetivorans. J Bacteriol 2010, 192:1511-1517. 31. Paytubi S, White MF: The crenarchaeal DNA damage-inducible transcription factor B paralogue TFB3 is a general activator of transcription. Mol Microbiol 2009, 72:1487-1499. This paper is the original report describing the increased expression of an alternative transcription factor (TFB3) upon UV-radiation in Sulfolobus solfataricus. This article has been used in this review to support the fact that Archaea can make use of alternative TFB/TBP combinations. 32. Hirtreiter A, Damsma GE, Cheung ACM, Klose D, Grohmann D, Vojnic E, Martin ACR, Cramer P, Werner F: Spt4/5 stimulates transcription elongation through the RNA polymerase clamp coiled-coil motif. Nucleic Acids Res 2010, 38:4040-4051. 33. Klein BJ, Bose D, Baker KJ, Yusoff ZM, Zhang X, Murakami KS: RNA polymerase and transcription elongation factor Spt4/5 complex structure. Proc Natl Acad Sci U S A 2011, 108:546-550. This paper presents the first structural investigation of the RNAP–Spt4/5 complex using recombinant proteins from Pyroccocus furiosus. Based on a low resolution cryo-EM density map a model for the Spt4/5 binding mode was built suggesting that Spt4/5 bridges over the DNA-binding channel. 34. Martinez-Rucobo FW, Sainsbury S, Cheung AC, Cramer P: Architecture of the RNA polymerase–Spt4/5 complex and basis of universal transcription processivity. EMBO J 2011 doi: 10.1038/emboj.2011.64. This publication provides the first crystal structure of an RNAP–Spt4/ 5NGN complex using the transcription system from Pyroccocus furiosus. A highlight of this paper is the idea to circumvent the RNAP–Spt4/5 crystallisation problems encountered so far by using an isolated and recombinant Rpo1 clamp domain that readily crystallised with Spt4/ 5NGN. The architecture of the full RNAP–Spt4/5 complex has been modelled and shown that Spt5–NGN domain closes the cleft over the active site to lock nucleic acids in the elongation complex. 35. Epshtein V, Nudler E: Cooperation between RNA polymerase molecules in transcription elongation. Science 2003, 300:801-805. 36. Saeki H, Svejstrup JQ: Stability, flexibility, and dynamic interactions of colliding RNA polymerase II elongation complexes. Mol Cell 2009, 35:191-205. 37. Santangelo TJ, Cubonova´ L, Matsumi R, Atomi H, Imanaka T, Reeve JN: Polarity in archaeal operon transcription in Thermococcus kodakaraensis. J Bacteriol 2008, 190:2244-2248. 38. Proshkin S, Rahmouni AR, Mironov A, Nudler E: Cooperation between translating ribosomes and RNA polymerase in transcription elongation. Science 2010, 328:504-508. 39. Burmann BM, Schweimer K, Luo X, Wahl MC, Stitt BL, Gottesman ME, Ro¨sch P: A NusE:NusG complex links transcription and translation. Science 2010, 328:501-504. 40. Santangelo TJ, Cubonova´ L, Skinner KM, Reeve JN: Archaeal intrinsic transcription termination in vivo. J Bacteriol 2009, 191:7102-7108. 41. Santangelo TJ, Reeve JN: Archaeal RNA polymerase is sensitive to intrinsic termination directed by transcribed and remote sequences. J Mol Biol 2006, 355:196-210.
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42. Spitalny P, Thomm M: A polymerase III-like reinitiation mechanism is operating in regulation of histone expression in archaea. Mol Microbiol 2008, 67:958-970. This is the first demonstration that archaeal RNAPs might employ an RNAPIII-like re-initiation mechanism. Template competition experiments revealed that efficiently terminated RNAPs initiate from the same template rather than binding to and starting from a promoter of an alternative template. 43. Sandman K, Reeve JN: Archaeal histones and the origin of the histone fold. Curr Opin Microbiol 2006, 9:520-525. 44. Hada K, Nakashima T, Osawa T, Shimada H, Kakuta Y, Kimura M: Crystal structure and functional analysis of an archaeal chromatin protein Alba from the hyperthermophilic archaeon Pyrococcus horikoshii OT3. Biosci Biotechnol Biochem 2008, 72:749-758. 45. Zhang Z, Gong Y, Guo L, Jiang T, Huang L: Structural insights into the interaction of the crenarchaeal chromatin protein Cren7 with DNA. Mol Microbiol 2010, 76:749-759. 46. Luo X, Schwarz-Linek U, Botting CH, Hensel R, Siebers B, White MF: CC1, a novel crenarchaeal DNA binding protein. J Bacteriol 2007, 189:403-409. 47. Guo L, Feng Y, Zhang Z, Yao H, Luo Y, Wang J, Huang L: Biochemical and structural characterization of Cren7, a novel chromatin protein conserved among Crenarchaea. Nucleic Acids Res 2008, 36:1129-1137. 48. Zhang Z, Pugh BF: High-resolution genome-wide mapping of the primary structure of chromatin. Cell 2011, 144:175-186. 49. Bailey KA, Marc F, Sandman K, Reeve JN: Both DNA and histone fold sequences contribute to archaeal nucleosome stability. J Biol Chem 2002, 277:9293-9301. 50. Wilkinson SP, Ouhammouch M, Geiduschek EP: Transcriptional activation in the context of repression mediated by archaeal histones. Proc Natl Acad Sci U S A 2010, 107:6777-6781. This report describes the effect of the activator Ptr2 counteracting histone-mediated transcriptional repression. The results presented suggest that the context of repressive chromatin may be a generally important component of archaeal gene regulation as discussed in this review. 51. Xie Y, Reeve JN: Transcription by an archaeal RNA polymerase is slowed but not blocked by an archaeal nucleosome. J Bacteriol 2004, 186:3492-3498. 52. Walther J, Sierocinski P, van der Oost J: Hot transcriptomics. Archaea 2011, 2010:897585. 53. Tachdjian S, Kelly RM: Dynamic metabolic adjustments and genome plasticity are implicated in the heat shock response of the extremely thermoacidophilic archaeon Sulfolobus solfataricus. J Bacteriol 2006, 188:4553-4559. 54. Koide T, Reiss DJ, Bare JC, Pang WL, Facciotti MT, Schmid AK, Pan M, Marzolf B, Van PT, Lo F et al.: Prevalence of transcription promoters within archaeal operons and coding sequences. Mol Syst Biol 2009, 5:285. 55. Furtwa¨ngler K, Tarasov V, Wende A, Schwarz C, Oesterhelt D: Regulation of phosphate uptake via Pst transporters in Halobacterium salinarum R1. Mol Microbiol 2010, 76:378-392. 56. Hausner W, Lange U, Musfeldt M: Transcription factor S, a cleavage induction factor of the archaeal RNA polymerase. J Biol Chem 2000, 275:12393-12399. 57. Ouhammouch M, Dewhurst RE, Hausner W, Thomm M, Geiduschek EP: Activation of archaeal transcription by recruitment of the TATA-binding protein. Proc Natl Acad Sci U S A 2003, 100:5097-5102. 58. Bell SD, Cairns SS, Robson RL, Jackson SP: Transcriptional regulation of an archaeal operon in vivo and in vitro. Mol Cell 1999, 4:971-982.
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Recent advances in the understanding of archaeal transcription Dina Grohmann and Finn Werner RNA polymerases (RNAPs) make repeatedly use of their templates by cycling through initiation, elongation and termination phases of transcription; during each step RNAP is interacting with and regulated by distinct transcription factors. The dynamic interplay between nucleic acid sequences, transcription factors and RNAP affects the activity and distribution of transcription complexes across the genome, and ultimately executes the genetic programme of the organism. This review covers recent discoveries about the mechanisms of archaeal transcription obtained by a combination of in vivo and in vitro approaches, from the molecular to the global level. Address RNAP Laboratory, Institute for Structural and Molecular Biology, Division of Biosciences, University College London, Darwin Building, Gower Street, London WC1E 6BT, United Kingdom Corresponding author: Grohmann, Dina ([email protected])
Current Opinion in Microbiology 2011, 14:328–334 This review comes from a themed issue on Archaea Edited by John Reeve and Christa Schleper Available online 17th May 2011 1369-5274/$ – see front matter # 2011 Elsevier Ltd. All rights reserved. DOI 10.1016/j.mib.2011.04.012
Introduction The last five years have witnessed the birth of archaeal genetics and systems biology and a combination of both with biochemical approaches has added to our understanding of differential gene expression in Archaea in response to environmental changes. Gene expression is controlled by the DNA sequence of the genome, by general and gene-specific transcription factors, by the RNAP and potentially by non-coding (nc) RNAs; Table 1 lists various mechanisms that are discussed in this review.
The RNAP transcription apparatus The archaeal transcription machinery is closely related to the eukaryotic systems in terms of RNAP structure and function, but relies on a minimal set of transcription factors and promoter elements (Figure 1a). Moreover, the molecular mechanisms accompanying the transcription cycle (Figure 1b) are conserved [1]. Archaeal RNAPs consist of 11–12 subunits that are structurally and functionally homologous to eukaryotic RNAPII subunits. The Current Opinion in Microbiology 2011, 14:328–334
core of the enzyme consists of the catalytic Rpo1 and Rpo2 subunits and an assembly platform made of Rpo3, Rpo10, Rpo11 and Rpo12. Rpo1 does not contain the Cterminal domain (CTD) common to its eukaryotic counterpart RBP1 and therefore lacks a target for many regulatory factors. A group of small RNAP subunits, which are not strictly required for basic RNAP function including promoter directed transcription, are incorporated into the enzyme close to the RNA exit channel (Rpo4/7), proximal to the NTP entry pore (Rpo8) and at the tip of the RNAP jaws (Rpo5 and Rpo13). Rpo4 and Rpo7 form the RNAP stalk, which is anchored to the RNAP via Rpo6 (Figure 2a). The stalk has emerged as an important feature of RNAP for all phases of the transcription cycle [2]. During initiation it promotes DNA melting and interacts with TFE [3–6]. During elongation the stalk interacts with the RNA transcript via an S1 motif in Rpo7 and not only facilitates robust processivity but also enhances transcription termination at weak termination signals [7]. The structure of the Rpo4/7 complex suggests that Rpo4 stabilises Rpo7 [8]. Genetic investigations have shown that whereas the archaeal rpo7 and the eukaryotic rpb7 are essential genes, archaeal rpo4 and eukaryotic rpb4 can be deleted with viability retained at moderate temperatures [6,9]. In addition to the RNAP stalk, mutations in the switch 3 motif in the Rpo2 subunit also affect the transcription elongation rate, possibly by promoting strand separation at the upstream edge of the DNA– RNA hybrid [10]. Phosphodiester bond formation and the mechanism of translocation that facilitates the movement of RNAP along the DNA template are facilitated by the active site. The RNAP bridge (Figure 2a, orange) and trigger helices are intimately involved in this process, and a saturation mutagenesis approach has provided exciting evidence that ‘superactive’ RNAP mutants correlate with distinct conformational states of the bridge helix [11,12].
Mechanisms of basal initiation factors Archaeal RNAP has a strict requirement for two basal factors, TBP (TATA binding protein) and TFB (transcription factor B) for transcription initiation [13,14] and highly conserved homologues of these factors, namely TBP and TFIIB, are required for eukaryotic transcription initiation. TBP binds to the TATA element of the promoter and distorts its topology by bending it approximately 908 [15]. The directionality of the promoter is brought forth by binding of TFB to the B-recognition element (BRE) upstream of the TATA element [16–18]. The TATA–TBP–TFB platform recruits RNAP forming the minimal preinitiation complex (PIC). In addition to recruiting RNAP, TFB contributes directly to the mechanism of initiation by donating a highly flexible linker www.sciencedirect.com
Understanding of archaeal transcription Grohmann and Werner 329
Table 1 Sequence elements and factors that regulate transcription in Archaea. The interplay between template DNA and transcript RNA sequence, basal and gene-specific transcription factors and the RNA polymerase governs the regulation of transcription. In addition histones and histone-like proteins and predicted non-coding RNAs including antisense transcripts have the potential to direct transcription in a gene-specific manner. Abbreviations: template strand (TS), non-template strand (NTS), B-recognition element (BRE), initiator element (Inr), tetrasome-localising sequence (TLS). The references are examples and not exhaustive. Nucleic acid element Initiation (promoter)
Elongation (intragenic)
Protein factor
TATA BRE
TBP TFB
TS at start site NTS at start site Inr/TS Pause (DNA) Pause (DNA) RNA non-sequence specific
Mechanism
Stimulation/ inhibition
Refs
+/ +/
[13,14] [13,16,17]
TFB TFE RNAP
TBP recruitment TFB and RNAP recruitment/directionality Initiation/catalysis DNA melting Promoter strength
? + +/
[5,19] [5,21] [24,25]
Spt4/5 TFS Rpo4/7
RNAP processivity Transcript cleavage RNAP processivity
+ + +
[32] [56] [7]
Termination (terminator)
poly-dA:rU signal
Rpo4/7
Enhanced termination
?
[7]
Gene-specific regulation
TATA/BRE
TBP/TFB, alternative variants Activator (e.g. Ptr2) Repressor (e.g. MDR1) Histones –
Promoter selection/internal promoters Ptr2: TBP recruitment MDR1: Promoter occlusion Promoter occlusion Hybridisation to mRNA
+/
[30]
+ ?
[57] [58] [49,50] [24,54]
Activator binding motif Repressor binding motif TLS antisense RNAs
region to complement the active site of RNAP proximal to the template strand (TS) [5,19,20]. A third archaeal initiation factor, TFE (transcription factor E), associates with RNAP and stimulates DNA melting (‘open complex formation’), during which the TS is loaded into the active site [5,21,22]. TFE interacts with the non-template strand (NTS) and thereby stabilises the PIC [21]. A recent computational search has identified a putative homologue of TFE beta (or C34, the RNAPIII homologue), but its function as initiation factor still needs to be validated experimentally [23]. In the open complex RNAP interacts with the TS around the transcription start site, which occasionally is referred to as the initiator element (Inr). The Inr has a weak sequence bias (mainly T(1)A(+1) [24]) and variations in its sequence affect the promoter strength, which suggests sequence-specific interactions between Inr and RNAP and/or TFB [5,25]. Many Archaea encode multiple homologues of the general transcription factors TBP and TFB. Halobacterium NRC-1 encodes six TBPs and seven TFB variants [26]. Theoretically this allows for 42 different TFB–TBP combinations that could facilitate differential regulation of transcription by alternative TFB–TBP combinations. In Halobacterium six out of 13 TBP and TFB genes are essential for growth under standard conditions [27]. ChIPchip results suggest that some of the TFB variants are enriched on a subset of promoters of genes with similar functions [28]. Ideally a combination of genetic and biochemical approaches should be used to dissect the contribution of the individual factors to gene-specific regulation. However, just a few organisms with estabwww.sciencedirect.com
lished in vitro transcription system are also genetically tractable with one excellent exception: Thermococcus kodakarensis. Thermococcus encodes two TFB variants, TFB1 and TFB2, and combined genetic and biochemical approaches have shown that both are active and support transcription from a range of promoters even though the optimal salt concentrations differ for the two variants in vitro [29]. However, the deletion of either TFB did not lead to any phenotypes in vivo, which suggests a high degree of redundancy. A deletion analysis of the three TBP homologues in Methanosarcina acetivorans revealed that just TBP1 is essential for survival and TBP2 and 3 play a role in environmental adaptation [30]. In addition to multiple isoforms of full length TBPs and TFBs, some Archaea also encode truncated versions of these transcription factors. For example, ultraviolet radiation of Sulfolobus solfataricus induces the expression of an unorthodox TFB variant, TFB3, which lacks the C-terminal core domain that is responsible for interactions with TBP and the promoter BRE sequence [31]. TFB3 is thus not able to form ternary DNA–TBP–TFB complexes. However, TFB3 competes with the canonical TFB1 for binding to RNAP, and interacts with the ternary TFB1– TBP–DNA complex. TFB3 stimulates transcription from several promoters including genes that are induced by UV irradiation. This stimulating effect is strictly dependent on TFB1, which suggests that TFB3 acts in concert with TFB1 on the same transcription initiation complexes by a mechanism that is not yet understood. Nevertheless, the cooperation of two TFB variants is a feature unique to the archaeal domain of life. Current Opinion in Microbiology 2011, 14:328–334
330 Archaea
Figure 1
RNAP subunits
(a)
Archaea
Eukaryotes
Rpo1 (A) Rpo2 (B) Rpo3 (D) Rpo11 (L) Rpo6 (K) Rpo5 (H) Rpo8* (G) Rpo10 (N) Rpo12 (P) Rpo4 (F) Rpo7 (E)
RPB1 RPB2 RPB3 RPB11 RPB6 RPB5 RPB8 RPB10 RPB12 RPB4 RPB7 RPB9
(b)
transcription factors
Termination
Initiation RNAP recruitment TBP/TFB
ribosome S10
Transcription cycle Initiation DNA melting TFE
Elongation Spt4/5 TFS
Rpo13* TBP TFB TFEα TFEβ/C34* TFS Spt4 Spt5 NusA
Re-initiation
TBP TFIIB TFIIEα TFIIEβ TFIIS Spt4 Spt5
Pause/backtrack/ arrest/termination? Initiation promoter escape
TFE
Spt4/5 Current Opinion in Microbiology
The archaeal transcription cycle. (a) Archaeal RNAP subunits and transcription factors and their eukaryotic RNAPII counterparts. Note that homologues of Rpo8, Rpo13 and TFEbeta/C34 are not present in all archaeal genomes (highlighted with asterisks). (b) RNAP repeatedly makes use of the same DNA template by cycling through initiation, elongation and termination phases. During initiation TBP and TFB assemble on the promoter and recruit RNAP. TFE stimulates the next stage of initiation during which the DNA is melted and the template strand loaded into the active site. Spt4/5 and TFS associate with the elongation complex and stimulate processivity; in addition Spt4/5 has the potential to couple transcription and translation via interactions between the Spt5 KOW domain and ribosomal protein S10 (NusE).
Post-initiation mechanisms of transcription Following promoter escape RNAP enters the elongation phase of transcription (Figure 1b). Elongation is a discontinuous process that is frequently interrupted by pausing, and transcription elongation factors alleviate these impediments. Spt5, a homologue of bacterial NusG, is a universally conserved elongation factor that usually interacts with Spt4 and, when bound to RNAP, increases transcription processivity [32]. The Spt5 NGN (NusGN-terminal) domain is required for this stimulatory activity and interacts with the RNAP clamp coiled coil [32] to form a bridge across the DNA-binding channel. Using the archaeal proteins it has been possible to obtain structural information of the RNAP–Spt4/5 complex for the first time [33,34]. These studies provide insights into the likely mechanism for the enhanced processivity of the RNAP upon interaction with Spt4/5. The structural investigations imply that Spt5 induces the closure of the clamp domain, which might lead to a further encapsulation of the DNA and thereby preventing the release of the DNA template and the dissociation of the elongation complex. The function of the archaeal Spt5 KOW (Kyprides–Ouzounis–Woese) domain is less clear, but in Bacteria the Current Opinion in Microbiology 2011, 14:328–334
NusG KOW domain recruits factors involved in termination/anti-termination mechanisms including the rho helicase and NusE. In Eukaryotes and Bacteria, multiple RNAPs transcribing the same operon/genein the same direction cooperate, most likely by preventing a retrograde movement of RNAP called back-tracking [35,36]. Similar to Bacteria, many archaeal genes are organised in multi-cistronic operons and mRNAs are translated co-transcriptionally. This gives rise to polarity — expression of a downstream gene is affected by genes upstream in the same operon [37]. In Bacteria the ratio of nucleotide and amino acid addition rates remains constant (between 2.9 and 3.1) over a range of addition and growth rates [38]. This adjusts the transcriptional yield to translational needs. A recent report suggests that NusG (Spt5) physically links elongating RNAPs and ribosomes via interactions between the NusG KOW domain and NusE, which is identical to ribosomal protein S10 [39]. This tethering of RNAP and ribosomes explains not only why ribosomes suppress sequence-dependent pausing of RNAP by ‘pushing’ them forward, but also why conditions that decrease translation rates (e.g. growth conditions, codon usage www.sciencedirect.com
Understanding of archaeal transcription Grohmann and Werner 331
Figure 2
(a)
(b) Ribosome 30S subunit structure
model stalk
mRNA transcript Rpo1 (A)
transcription
Rpo2 (B)
clamp
Rpo3 (D)
NusE(S10)
Rpo11 (L)
Spt4
Rpo6 (K)
Spt5 NGN Spt5 KOW
Rpo4 (F) Rpo5 (H)
duplex DNA
RNAP
Rpo7 (E) Rpo8 (G)
3’
Rpo10 (N) Rpo12 (P)
assembly platform catalytic centre
Rpo13
bridge helix
downstream DNA binding site
catalytic centre Current Opinion in Microbiology
Structure and function of RNAP. (a) Important features of RNAP that are discussed in the text are circled in red (assembly platform), blue (stalk), green (clamp) and firebrick red (active site). The downstream duplex DNA is indicated as grey cylinder. RNAP structure is based on Sulfolobus (pdb 2WAQ), the RNAP subunits are colour-coded according to convention [1]. (b) Working model of the Spt4/5 modus operandi. The universally conserved elongation factor Spt4/5 (NusG in bacteria) is recruited to the RNAP clamp coiled coil and closes the DNA-binding channel over the DNA template (duplex DNA indicated as salmon pink double helix, RNA in red). This interaction may deny the dissociation of the template from the elongation complex and thereby enhance processivity. In Bacteria, and likely in Archaea, the NusG KOW domain (green) interacts with ribosomal protein S10 (blue), which tethers elongating ribosomes (30S subunit shown in firebrick red, not drawn to scale) and RNAP.
and antibiotics) also lower transcription elongation rates [38]. Based on the high evolutionary conservation of the Spt5 and NusG KOW domains and S10 it is likely that this mechanism also operates in Archaea (Figure 2b). Transcription termination is the final and least well understood phase of the transcription cycle. Archaeal RNAP appears to terminate via a mechanism similar to that employed by eukaryotic RNAPIII in which short poly-dA stretches in the template DNA (resulting in rU:dA RNA:DNA hybrids) serve as signals for efficient termination in vivo and in vitro, without any requirements for secondary structures in the transcript (such as hairpins), or exogenous termination factors (e.g. the bacterial rho factor) [7,40,41]. The transcription elongation complex is very stable and any model for transcription termination must provide a convincing rationale for its destabilisation and dissociation. This is likely to involve an opening of the RNAP clamp and requires the dissociation of any RNAP-bound Spt4/5 from the elongation complex. Alternatively, recent data suggest that terminated archaeal RNAPs are ‘recycled’ to the promoter and www.sciencedirect.com
re-initiate transcription like the eukaryotic RNAPIII enzyme [42].
Regulation of transcription by chromatin Different Archaea encode many positively charged but evolutionary unrelated DNA-binding proteins including histones [43], Alba [44], Sac7d [45], CC1 [46] and Cren7 [47], which are thought to compact the archaeal DNA in chromatin-like structures. Archaeal histones are smaller than their eukaryotic homologues, but adopt the typical histone fold that is formed by three alpha-helices separated by two short beta-strand loops. They lack the tails that are extensively post-translational modified in eukaryotic histones [48]. While Eukaryotes compact their nuclear DNA employing exclusively the same four histones Archaea often encode more than one histone variant that readily form homodimers and heterodimers. These can assemble into tetramers in the presence of DNA and compact approximately 80 bp of DNA, as compared to the octameric histones that compact 147 bp in Eukaryotes. Even though strict nucleosome positioning sequences have not been identified in Current Opinion in Microbiology 2011, 14:328–334
332 Archaea
Archaea yet, a SELEX approach has identified DNA sequences that preferentially are bound by archaeal histones (tetrasome-localising sequences, TLSs) [49]. This result makes it plausible that sequence-specific chromatin-assemblies can regulate transcription by allowing/denying access of the transcription machinery to the promoters [49,50]. Archaeal histone binding to a template DNA reduces but does not fully inhibit promoterdependent transcription in vitro, except at non-physiologically high histone:DNA ratios. However, the histonemediated transcriptional repression can be relieved in vitro by at least one activator, designated Ptr2 [50]. Most likely Ptr2 competes with histones for the binding to the promoter DNA and, when bound, allows RNAP and basal transcription factors access to the template. Archaeal RNAPs are able to elongate through histone-covered DNA templates, albeit at reduced rates [51]. In summary, the competition between histones, transcriptional activators and transcription initiation factors as shown in in vitro experiments has the potential to orchestrate gene-specific regulation of transcription also in vivo (Figure 3).
Global approaches to archaeal gene expression The prediction of operon organisation is problematic at best, but the deep sequencing of the S. solfataricus transcriptome proved beyond doubt that protein encoding genes in Archaea are organised both in mono-cistronic (72%) and poly-cistronic (28%) operons [24]. Early archaeal transcriptomic experiments used microarray technology and aimed at the detection of changes in gene expression under different growth conditions and environmental insults (e.g. heat shock, osmotic shock, viral infection, for a recent review [52]). In S. solfataricus the operon encoding the large Rpo1 and Rpo2 subunits is down regulated under heat shock conditions, which serves as a fast global shutdown of transcription in response to severe stress [53]. Archaea encode a plethora of ncRNAs. In Sulfolobus at least 310 ncRNAs have been identified, some of which have the potential to regulate gene expression both at the transcriptional and posttranscriptional level. The majority of ncRNAs identified in transcriptomic studies are antisense transcripts of existing operons (Figure 3). A closer scrutiny revealed that these operons primarily encode genes involved in ion transport and metabolism [24]. Microarray transcriptomics in Halobacterium combined with whole genome occupancy profiling (ChIP-chip) showed that Archaea make use of alternative intragenic promoters in a growth phase-dependent manner [54]. Ten percent of the TBP/ TFB transcription factor binding sites map to the coding regions of multi-cistronic operons, and appear to act as additional transcription start sites (Figure 3). Conditional transcription of poly-cistronic operons has been described for the sdhCDBA operon. Whereas sdhCDB (encoding for Current Opinion in Microbiology 2011, 14:328–334
Figure 3
(a)
Internal promoter in a polycistronic unit A
B
C
D
(b)
Antisense transcription
(c)
Regulation by multiple TFB/TBP combinations TBP1/TFB1
(d)
TBP1/TFB3
TBP4/TFB1
Promoter occlusion by histones
X Ptr2 Ptr2
Histone-like protein Current Opinion in Microbiology
Transcriptional regulation. Archaeal poly-cistronic operons have alternative internal promoters (highlighted with a dotted blue arrow) that are subjected to growth phase-dependent regulation (a). Antisense transcripts are synthesised from promoters on the complementary strand of the ORF; they can potentially anneal with the mRNA by Watson–Crick basepairing and affect its translation and/or stability (b). Combinations of TBP and TFB variants may recruit RNAP to subsets of genes and thereby regulate transcription in a gene-specific fashion in archaeal organisms that encode multiple TBP/TFB variants (c). Histonelike proteins have been shown to occlude promoter elements and repress transcription; transcription activators such as Ptr2 can activate transcription by competing with histones for promoter binding (d).
subunits of the succinate-dehydrogenease (SDH) enzyme complex, the Fe–S, cytochrome b and membrane anchor subunits) is down regulated at high cell densities, the expression of sdhA (encoding for the FAD-binding flavoprotein component) remains unchanged. And recent studies on the pst (phosphate-specific transport) operon in Halobacterium salinarum support the finding that Archaea make use of alternative promoters [55]. Here, transcription starts at two different promoters within the multi-cistronic operon in a phosphate-dependent manner. In summary, the transcription of archaeal poly-cistronic operons is fine-tuned and alternative internal promoters are either induced or repressed depending upon environmental conditions.
Perspective Many of the discoveries described in this review add to our understanding about transcriptional regulation in www.sciencedirect.com
Understanding of archaeal transcription Grohmann and Werner 333
Archaea even though some mechanistic details are not elucidated yet. For example the meaning of multiple copies of the transcription factors TFB and TBP encoded in some archaeal organisms remain unclear despite the advances in archaeal genetics.
11. Tan L, Wiesler S, Trzaska D, Carney HC, Weinzierl ROJ: Bridge helix and trigger loop perturbations generate superactive RNA polymerases. J Biol 2008, 7:40. 12. Weinzierl ROJ: Nanomechanical constraints acting on the catalytic site of cellular RNA polymerases. Biochem Soc Trans 2010, 38:428-432. 13. Werner F, Weinzierl ROJ: A recombinant RNA polymerase II-like enzyme capable of promoter-specific transcription. Mol Cell 2002, 10:635-646.
The challenge for the future will be to validate the wealth of information derived from systems biology approaches and to observe the archaeal RNAP encountering ‘roadblocks’ like histones in vivo. This includes for instance, firstly, a detailed description of the molecular mechanisms that couple transcription and translation in Archaea, secondly, to determine whether or not archaeal genomes are compacted by histones and histone-like proteins in vivo and thirdly to specify the function of the ncRNAs identified in transcriptomic studies.
15. Grohmann D, Klose D, Fielden D, Werner F: FRET (fluorescence resonance energy transfer) sheds light on transcription. Biochem Soc Trans 2011, 39:122-127.
Acknowledgements
17. Bell SD, Kosa PL, Sigler PB, Jackson SP: Orientation of the transcription preinitiation complex in archaea. Proc Natl Acad Sci U S A 1999, 96:13662-13667.
Research at the RNAP Laboratory was funded by project grants from the Wellcome Trust (079351/Z/06/Z) and BBSRC (BB/E008232/1 and BB/ H019332/1) to Finn Werner. We apologise to our colleagues whose work could not be cited due to space constraints.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
Werner F, Grohmann D: Evolution of multisubunit RNA polymerases in the three domains of life. Nat Rev Microbiol 2011, 9:85-98.
2.
Grohmann D, Werner F: Cycling through transcription with the RNA polymerase F/E (RPB4/7) complex: structure, function and evolution of archaeal RNA polymerase. Res Microbiol 2011, 162:10-18.
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Naji S, Gru¨nberg S, Thomm M: The RPB7 orthologue E0 is required for transcriptional activity of a reconstituted archaeal core enzyme at low temperatures and stimulates open complex formation. J Biol Chem 2007, 282:11047-11057. Ouhammouch M, Werner F, Weinzierl ROJ, Geiduschek EP: A fully recombinant system for activator-dependent archaeal transcription. J Biol Chem 2004, 279:51719-51721.
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