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Archaea are a key feature of the terran biosphere. Since their early characterization in the 1970s, we have learned much about the fascinating organism, most recently by applying genome-, transcriptome-, proteome-, and metabolome-scale methods. This chapter describes seminal contributions that elaborate on the study of archaeal biology at the systems level.

Transcription is enabled by RNA polymerase and general factors that allow its progress through the transcription cycle by facilitating initiation, elongation and termination. The transitions between specific stages of the transcription cycle provide opportunities for the global and gene-specific regulation of gene expression. The exact mechanisms and the extent to which the different steps of transcription are exploited for regulation vary between the domains of life, individual species and transcription units. However, a surprising degree of conservation is apparent. Similar key steps in the transcription cycle can be targeted by homologous or unrelated factors providing insights into the mechanisms of RNAP and the evolution of the transcription machinery. Archaea are bona fide prokaryotes but employ a eukaryote-like transcription system to express the information of bacteria-like genomes. Thus, archaea provide the means not only to study transcription mechanisms of interesting model systems but also to test key concepts of regulation in this arena. In this review, we discuss key principles of archaeal transcription, new questions that still await experimental investigation, and how novel integrative approaches hold great promise to fill this gap in our knowledge.

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.

Summary Eukaryotic TIP49a (Pontin) and TIP49b (Reptin) AAA+ ATPases play essential roles in key cellular processes. How their weak ATPase activity contributes to their important functions remains largely unknown and difficult to analyze because of the divergent properties of TIP49a and TIP49b proteins and of their homo- and hetero-oligomeric assemblies. To circumvent these complexities, we have analyzed the single ancient TIP49 ortholog found in the archaeon Methanopyrus kandleri (mkTIP49). All-atom homology modeling and molecular dynamics simulations validated by biochemical assays reveal highly conserved organizational principles and identify key residues for ATP hydrolysis. An unanticipated crosstalk between Walker B and Sensor I motifs impacts the dynamics of water molecules and highlights a critical role of trans-acting aspartates in the lytic water activation step that is essential for the associative mechanism of ATP hydrolysis., Graphical Abstract, Highlights • We have studied the single TIP49 ortholog (mkTIP49) from the archaeon M. kandleri • We propose a model for assembly of the pre-transition state for ATP hydrolysis • Trans-aspartates downregulate ATP hydrolysis by mkTIP49 hexamers • Mutational analysis confirms a highly conserved mechanism for lytic water activation, Afanasyeva et al. combine computational and biochemical analysis to reveal conserved organizational principles and residues critical for ATP hydrolysis in the AAA+ ATPase TIP49 ortholog from archaea. They highlight a role of trans-aspartates in the lytic water activation step essential for ATP hydrolysis.

Transcription is an intrinsically dynamic process and requires the coordinated interplay of RNA polymerases (RNAPs) with nucleic acids and transcription factors. Classical structural biology techniques have revealed detailed snapshots of a subset of conformational states of the RNAP as they exist in crystals. A detailed view of the conformational space sampled by the RNAP and the molecular mechanisms of the basal transcription factors E (TFE) and Spt4/5 through conformational constraints has remained elusive. We monitored the conformational changes of the flexible clamp of the RNAP by combining a fluorescently labeled recombinant 12-subunit RNAP system with single-molecule FRET measurements. We measured and compared the distances across the DNA binding channel of the archaeal RNAP. Our results show that the transition of the closed to the open initiation complex, which occurs concomitant with DNA melting, is coordinated with an opening of the RNAP clamp that is stimulated by TFE. We show that the clamp in elongation complexes is modulated by the nontemplate strand and by the processivity factor Spt4/5, both of which stimulate transcription processivity. Taken together, our results reveal an intricate network of interactions within transcription complexes between RNAP, transcription factors, and nucleic acids that allosterically modulate the RNAP during the transcription cycle.

Evolutionary related multisubunit RNA polymerases (RNAPs) transcribe the genomes of all living organisms. Whereas the core subunits of RNAPs are universally conserved in all three domains of life—indicative of a common evolutionary descent—this only applies to one RNAP-associated transcription factor—Spt5, also known as NusG in bacteria. All other factors that aid RNAP during the transcription cycle are specific for the individual domain or only conserved between archaea and eukaryotes. Spt5 and its bacterial homologue NusG regulate gene expression in several ways by (i) modulating transcription processivity and promoter proximal pausing, (ii) coupling transcription and RNA processing or translation, and (iii) recruiting termination factors and thereby silencing laterally transferred DNA and protecting the genome against double-stranded DNA breaks. This review discusses recent discoveries that identify Spt5-like factors as evolutionary conserved nexus for the regulation and coordination of the machineries responsible for information processing in the cell., Graphical Abstract Highlights ► Spt5–NusG is the only universally conserved transcription factor in the three domains of life. ► Highly versatile multifunction factor. ► Core function is to enhance transcription processivity. ► KOW domains serve as recruitment platform for broad range of factors. ► Instrumental in promoter proximal stalling in eukaryotes. ► Recruits chromatin remodelling and RNA processing factors in eukaryotes. ► Couples transcription and translation in prokaryotes. ► Recruits rho termination factor in bacteria.

Summary TFIIE and the archaeal homolog TFE enhance DNA strand separation of eukaryotic RNAPII and the archaeal RNAP during transcription initiation by an unknown mechanism. We have developed a fluorescently labeled recombinant M. jannaschii RNAP system to probe the archaeal transcription initiation complex, consisting of promoter DNA, TBP, TFB, TFE, and RNAP. We have localized the position of the TFE winged helix (WH) and Zinc ribbon (ZR) domains on the RNAP using single-molecule FRET. The interaction sites of the TFE WH domain and the transcription elongation factor Spt4/5 overlap, and both factors compete for RNAP binding. Binding of Spt4/5 to RNAP represses promoter-directed transcription in the absence of TFE, which alleviates this effect by displacing Spt4/5 from RNAP. During elongation, Spt4/5 can displace TFE from the RNAP elongation complex and stimulate processivity. Our results identify the RNAP “clamp” region as a regulatory hot spot for both transcription initiation and transcription elongation., Graphical Abstract Highlights ► The RNAP clamp coiled coil and RNAP stalk are required for TFE binding and activity ► The elongation factor Spt4/5 can inhibit PIC formation and transcription initiation ► TFE efficiently prevents inhibition of transcription initiation by Spt4/5 ► Spt4/5 displaces TFE from the TEC

Spt5 is the only known RNA polymerase-associated factor that is conserved in all three domains of life. We have solved the structure of the Methanococcus jannaschii Spt4/5 complex by X-ray crystallography, and characterized its function and interaction with the archaeal RNAP in a wholly recombinant in vitro transcription system. Archaeal Spt4 and Spt5 form a stable complex that associates with RNAP independently of the DNA-RNA scaffold of the elongation complex. The association of Spt4/5 with RNAP results in a stimulation of transcription processivity, both in the absence and the presence of the non-template strand. A domain deletion analysis reveals the molecular anatomy of Spt4/5--the Spt5 Nus-G N-terminal (NGN) domain is the effector domain of the complex that both mediates the interaction with RNAP and is essential for its elongation activity. Using a mutagenesis approach, we have identified a hydrophobic pocket on the Spt5 NGN domain as binding site for RNAP, and reciprocally the RNAP clamp coiled-coil motif as binding site for Spt4/5.

Archaeal and eukaryotic RNAPs (DNA-dependent RNA polymerases) are complex multi-subunit enzymes. Two of the subunits, F and E, which together form the F/E complex, have been hypothesized to associate with RNAP in a reversible manner during the transcription cycle. We have characterized the molecular interactions between the F/E complex and the RNAP core. F/E binds to RNAP with submicromolar affinity and is not in a dynamic exchange with unbound F/E.

RNA polymerases (RNAPs) are essential to all life forms and therefore deserve our special attention. The archaeal RNAP is closely related to eukaryotic RNAPII in terms of subunit composition and architecture, promoter elements and basal transcription factors required for the initiation and elongation phase of transcription. RNAPs of this class are large and sophisticated enzymes that interact in a complex manner with DNA/RNA scaffolds, substrates NTPs and a plethora of transcription factors - interactions that often result in an allosteric regulation of RNAP activity. The 12 subunits of RNAP play distinct roles including RNAP assembly and stability, catalysis and functional contacts with exogenous factors. Due to the availability of structural information of RNAPs at high-resolution and wholly recombinant archaeal transcription systems, we are beginning to understand the molecular mechanisms of archaeal RNAPs and transcription in great detail.

The transcriptional apparatus is one of the most complex cellular machineries and in order to fully appreciate the behavior of these protein–nucleic acid assemblies one has to understand the molecular details of the system. In addition to classical biochemical and structural studies, fluorescence-based techniques turned out as an important – and sometimes the critical – tool to obtain information about the molecular mechanisms of transcription. Fluorescence is not only a multi-modal parameter that can report on molecular interactions, environment and oligomerization status. Measured on the single-molecule level it also informs about the heterogeneity of the system and gives access to distances and distance changes in the molecular relevant nanometer regime. A pre-requisite for fluorescence-based measurements is the site-specific incorporation of one or multiple fluorescent dyes. In this respect, the archaeal transcription system is ideally suited as it is available in a fully recombinant form and thus allows for site-specific modification via sophisticated labeling schemes. The application of fluorescence based approaches to the archaeal transcription apparatus changed our understanding of the molecular mechanisms and dynamics that drive archaeal transcription and unraveled the architecture of transcriptional complexes not amenable to structural interrogation.

Archaeal RNA polymerase and general transcription factors are more closely related to those of eukaryotes than of bacteria. As such the study of transcription of archaea is important both in terms of examination of the evolution of the transcriptional machinery and as a simplified tool for eukaryotic transcription. In particular, the hyperthermophilic Methanocaldococcus jannaschii provides us with a fully recombinant RNA polymerase system allowing for much more detailed in vitro examination of the roles of different components during the transcription cycle than otherwise possible. The individual subunits of M. jannaschii enzyme are easily expressed and purified from heterologous expression systems. Forming functional RNA polymerase involves simply combining the different subunits under denaturing conditions and slowly removing the denaturant.

The molecular architecture of RNAPII-like transcription initiation complexes has been studied for years but its structure has remained opaque due to its conformational flexibility and size. We determined the three-dimensional architecture of the complete open complex (OC) composed of the promoter DNA, TATA box-binding protein (TBP), transcription factors TFB and TFE, and the 12-subunit RNA polymerase (RNAP) from M. jannaschii. By combining single-molecule Förster resonance energy transfer (smFRET) and the Bayesian parameter estimation based Nano-Positioning System (NPS) analysis, we modelled the entire archaeal OC, which elucidates the path of the ntDNA strand and interaction sites of the transcription factors with the RNAP. Compared to models of the eukaryotic OC, the position of the TATA DNA region with TBP and TFB is positioned closer to the surface of the RNAP, likely providing the mechanism by which DNA melting can occur in a minimal factor configuration, without the dedicated translocase/helicase encoding factor TFIIH.

The fully recombinant Methanocaldococcus jannaschii RNA polymerase allows for a detailed dissection of the different stages of the transcription. In the previous chapter, we discussed how to purify the different components of the M. jannaschii transcription system, the RNA polymerase subunits, and general transcription factors and how to assemble a functional M. jannaschii enzyme. Standard in vitro transcription assays can be used to examine the different stages of transcription. In this chapter, we describe how some of these assays have been optimized for M. jannaschii RNA polymerase, which transcribes at much higher temperatures than many other transcription complexes.

The Rpb4 and Rpb7 subunits of eukaryotic RNA polymerase II (RNAP(II)) form a heterodimer that protrudes from the 10-subunit core of the enzyme. We have obtained crystals of the human Rpb4/Rpb7 heterodimer and determined the structure to 2.7 A resolution. The presence of putative RNA-binding domains on the Rpb7 subunit and the position of the heterodimer close to the RNA exit groove in the 12 subunit yeast polymerase complex strongly suggests a role for the heterodimer in binding and stabilizing the nascent RNA transcript. We have complemented the structural analysis with biochemical studies directed at dissecting the RNA-binding properties of the human Rpb4/Rpb7 complex and that of the homologous E/F complex from Methanocaldococcus jannaschii. A number of conserved, solvent-exposed residues in both the human Rpb7 subunit and the archaeal E subunit have been modified by site-directed mutagenesis and the mutants tested for RNA binding by performing electrophoretic mobility shift assays. These studies have identified an elongated surface region on the corresponding face of both subunit E and Rpb7 that is involved in RNA binding. The area spans the nucleic acid binding face of the OB fold, including the B4-B5 loop, but also extends towards the N-terminal domain.

The high affinity interaction between the urokinase-type plasminogen activator (uPA) and its glycolipid-anchored cellular receptor (uPAR) promotes plasminogen activation and the efficient generation of pericellular proteolytic activity. We demonstrate here that expression of the tetraspanin CD82/KAI1 (a tumor metastasis suppressor) leads to a profound effect on uPAR function. Pericellular plasminogen activation was reduced by approximately 50-fold in the presence of CD82, although levels of components of the plasminogen activation system were unchanged. uPAR was present on the cell surface and molecularly intact, but radioligand binding analysis with uPA and anti-uPAR antibodies revealed that it was in a previously undetected cryptic form unable to bind uPA. This was not due to direct interactions between uPAR and CD82, as they neither co-localized on the cell surface nor could be co-immunoprecipitated. However, expression of CD82 led to a redistribution of uPAR to focal adhesions, where it was shown by double immunofluorescence labeling to co-localize with the integrin alpha(5)beta(1), which was also redistributed in the presence of CD82. Co-immunoprecipitation experiments showed that, in the presence of CD82, uPAR preferentially formed stable associations with alpha(5)beta(1), but not with a variety of other integrins, including alpha(3)beta(1). These data suggest that CD82 inhibits the proteolytic function of uPAR indirectly, directing uPAR and alpha(5)beta(1) to focal adhesions and promoting their association with a resultant loss of uPA binding. This represents a novel mechanism whereby tetraspanins, integrins, and uPAR, systems involved in cell adhesion and migration, cooperate to regulate pericellular proteolytic activity and may suggest a mechanism for the tumor-suppressive effects of CD82/KAI1.

Human vascular smooth muscle cells (VSMC) bind tissue plasminogen activator (tPA) specifically, saturably, and with relatively high affinity (K(d) 25 nM), and this binding potentiates the activation of cell-associated plasminogen (Ellis, V., and Whawell, S. A. (1997) Blood 90, 2312-2322). We have observed that this binding can be efficiently competed by DFP-inactivated tPA and S478A-tPA but not by tPA inactivated with H-D-Phe-Pro-Arg-chloromethyl ketone (PPACK). VSMC-bound tPA also exhibited a markedly reduced inhibition by PPACK, displaying biphasic kinetics with second-order rate constants of 7. 5 x 10(3) M(-1) s(-1) and 0.48 x 10(3) M(-1) s(-1), compared with 7. 2 x 10(3) M(-1) s(-1) in the solution phase. By contrast, tPA binding to fibrin was competed equally well by all forms of tPA, and its inhibition was unaltered. These effects were shown to extend to the physiological tPA inhibitor, plasminogen activator inhibitor 1. tPA.plasminogen activator inhibitor 1 complex did not compete tPA binding to VSMC, and the inhibition of bound tPA was reduced by 30-fold. The behavior of the various forms of tPA bound to VSMC correlated with conformational changes in tPA detected by CD spectroscopy. These data suggest that tPA binds to its specific high affinity site on VSMC by a novel mechanism involving the serine protease domain of tPA and distinct from its binding to fibrin. Furthermore, reciprocally linked conformational changes in tPA appear to have functionally significant effects on both the interaction of tPA with its VSMC binding site and the susceptibility of bound tPA to inhibition.

The core components of the archaeal transcription apparatus closely resemble those of eukaryotic RNA polymerase II, while the DNA-binding transcriptional regulators are predominantly of bacterial type. Here we report the construction of an entirely recombinant system for positively regulated archaeal transcription. By omitting individual subunits, or sets of subunits, from the in vitro assembly of the 12-subunit RNA polymerase from the hyperthermophile Methanocaldococcus jannaschii, we describe a functional dissection of this RNA polymerase II-like enzyme, and its interactions with the general transcription factor TFE, as well as with the transcriptional activator Ptr2.

RNA polymerases (RNAPs) from the three domains of life, Bacteria, Archaea and Eukarya, are evolutionarily related and thus have common structural and functional features. Despite the radically different morphology of Archaea and Eukarya, their RNAP subunit composition and utilisation of basal transcription factors are almost identical. This review focuses on the multiple functions of the most prominent feature that differentiates these enzymes from the bacterial RNAP--a stalk-like protrusion, which consists of the heterodimeric F/E subcomplex. F/E is highly versatile, it facilitates DNA strand-separation during transcription initiation, increases processivity during the elongation phase of transcription and ensures efficient transcription termination.

RNA polymerases (RNAP) carry out transcription, the first step in the highly regulated process of gene expression. RNAPs are complex multisubunit enzymes, which undergo extensive structural rearrangements during the transcription cycle (initiation-elongation-termination). They accommodate interactions with the nucleic acid scaffold of transcription complexes (template DNA, DNA/RNA hybrid, and nascent RNA) and interact with a plethora of transcription factors. Here we focused on the RNAP-F/E subcomplex, which forms a stable heterodimer that binds the nascent RNA and thereby stimulates the processivity of elongation complexes. We used the pulsed-EPR method DEER and fluorescence spectroscopy to probe for conformational changes within the F/E dimer. Our results demonstrate that, upon binding of RNA, F/E remains in a stable conformation, which suggests that it serves as a structurally rigid guiding rail for the growing RNA chain during transcription.

Evolutionary related multisubunit RNA polymerases from all three domains of life, Eukarya, Archaea and Bacteria, have common structural and functional properties. We have recently shown that two RNAP subunits, F/E (RPB4/7)—which are conserved between eukaryotes and Archaea but have no bacterial homologues—interact with the nascent RNA chain and thereby profoundly modulate RNAP activity. Overall F/E increases transcription processivity, but it also stimulates transcription termination in a sequence-dependent manner. In addition to RNA-binding, these two apparently opposed processes are likely to involve an allosteric mechanism of the RNAP clamp. Spt4/5 is the only known RNAP-associated transcription factor that is conserved in all three domains of life, and it stimulates elongation similar to RNAP subunits F/E. Spt4/5 enhances processivity in a fashion that is independent of the nontemplate DNA strand, by interacting with the RNAP clamp. Whereas the molecular mechanism of Spt4/5 is universally conserved in evolution, the added functionality of F/E-like complexes has emerged after the split of the bacterial and archaeo-eukaryotic lineages. Interestingly, bacteriophage-encoded antiterminator proteins could, in theory, fulfil an analogous function in the bacterial RNAP.

Archaeal RNA polymerases (RNAPs) are recruited to promoters through the joint action of three basal transcription factors: TATA-binding protein, TFB (archaeal homolog of TFIIB), and TFE (archaeal homolog of TFIIE). Our results demonstrate several new insights into the mechanisms of TFB and TFE during the transcription cycle. (i) The N-terminal Zn ribbon of TFB displays a surprising degree of redundancy for the recruitment of RNAP during transcription initiation in the archaeal system. (ii) The B-finger domain of TFB participates in transcription initiation events by stimulating abortive and productive transcription in a recruitment-independent function. TFB thus combines physical recruitment of the RNAP with an active role in influencing the catalytic properties of RNAP during transcription initiation. (iii) TFB mutations are complemented by TFE, thereby demonstrating that both factors act synergistically during transcription initiation. (iv) An additional function of TFE is to dynamically alter the nucleic acid-binding properties of RNAP by stabilizing the initiation complex and destabilizing elongation complexes.

We have demonstrated that tissue plasminogen activator (tPA) binds specifically to human vascular smooth muscle cells (VSMC) in a functionally relevant manner, both increasing plasminogen activation and decreasing tPA inhibition (Ellis, V., and Whawell, S. A. (1997) Blood 90, 2312-2322; Werner, F., Razzaq, T. M., and Ellis, V. (1999) J. Biol. Chem. 274, 21555-21561). To further understand this system we have now identified and characterized the protein responsible for this binding. Rat VSMC were surface-labeled with 125I, and cell lysates were subjected to an affinity chromatography scheme based on the previously identified tPA binding characteristics. A single radiolabeled protein of 63 kDa bound specifically and was eluted at low pH. This protein was isolated from large scale preparations of VSMC and unambiguously identified as the rat homologue of the human type-II transmembrane protein p63 (CKAP4) by matrix-assisted laser desorption ionization and nano-electrospray tandem mass spectrometry of tryptic fragments. In confirmation of this, a monoclonal antibody raised against authentic human p63 recognized the isolated protein in Western blotting. Immunofluorescence microscopy demonstrated that p63 was located principally in the endoplasmic reticulum but was also detected in significant quantities on the surface of human VSMC. In support of the hypothesis that p63 is the functional tPA binding site on VSMC, an anti-p63 monoclonal antibody was found to block tPA binding. Furthermore, heterologous expression of an N-terminally truncated mutant of p63, which targets exclusively to the plasma membrane, led to an increase in tPA-catalyzed plasminogen activation. Therefore, p63 on the surface of VSMC may contribute to the functional regulation of the plasminogen activation system in the vessel wall.

In the archaeal RNA polymerase and the eukaryotic RNA polymerase II, two subunits (E/F and RPB4/RPB7, respectively) form a heterodimer that reversibly associates with the core of the enzyme. Recently it has emerged that this heterodimer also has a counterpart in the other eukaryotic RNA polymerases: in particular two subunits of RNA polymerase I (A14 and A43) display genetic and biochemical characteristics that are similar to those of the RPB4 and RPB7 subunits, despite the fact that only A43 shows some sequence homology to RPB7. We demonstrate that the sequence of A14 strongly suggests the presence of a HRDC domain, a motif that is found at the C-terminus of a number of helicases and RNases. The same motif is also seen in the structure of the F subunit, suggesting a structural link between A14 and the RPB4/C17/subunit F family, even in the absence of direct sequence homology. We show that it is possible to co-express and co-purify large amounts of the recombinant A14/A43 heterodimer, indicating a tight and specific interaction between the two subunits. To shed light on the function of the heterodimer, we performed gel mobility shift assays and showed that the A14/A43 heterodimer binds single-stranded RNA in a similar way to the archaeal E/F complex.

RNA polymerases (RNAPs) are core components of the cellular transcriptional machinery. Progress with functional studies of eukaryotic RNAPs has been delayed by the fact that it has not yet been possible to assemble active enzymes from individual subunits. Archaeal RNAPs are directly comparable to eukaryotic RNAPII in terms of primary sequence homology and quaternary structure. Here we report the successful in vitro assembly of a recombinant archaeal RNAP from purified subunits. The recombinant enzyme displays full activity in transcription assays and is capable, in the presence of two other basal factors, of promoter-specific transcription. The assembly of mutant enzymes yielded several unexpected insights into the structural and functional contributions of various subunits toward overall RNAP activity.

The eukaryotic subunits RPB4 and RPB7 form a heterodimer that reversibly associates with the RNA polymerase II core and constitute the only two components of the enzyme for which no structural information is available. We have determined the crystal structure of the complex between the Methanococcus jannaschii subunits E and F, the archaeal homologs of RPB7 and RPB4. Subunit E has an elongated two-domain structure and contains two potential RNA binding motifs, while the smaller F subunit wraps around one side of subunit E, at the interface between the two domains. We propose a model for the interaction between RPB4/RPB7 and the core RNA polymerase in which the RNA binding face of RPB7 is positioned to interact with the nascent RNA transcript.