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African swine fever virus (ASFV) has a major global economic impact. With a case fatality in domestic pigs approaching 100%, it currently presents the largest threat to animal farming. Although genomic differences between attenuated and highly virulent ASFV strains have been identified, the molecular determinants for virulence at the level of gene expression have remained opaque. Here we characterise the transcriptome of ASFV genotype II Georgia 2007/1 (GRG) during infection of the physiologically relevant host cells, porcine macrophages. In this study we applied Cap Analysis Gene Expression sequencing (CAGE-seq) to map the 5’ ends of viral mRNAs at 5 and 16 hours post-infection. A bioinformatics analysis of the sequence context surrounding the transcription start sites (TSSs) enabled us to characterise the global early and late promoter landscape of GRG. We compared transcriptome maps of the GRG isolate and the lab-attenuated BA71V strain that highlighted GRG virulent-specific transcripts belonging to multigene families, including two predicted MGF 100 genes I7L and I8L. In parallel, we monitored transcriptome changes in the infected host macrophage cells. Of the 9,384 macrophage genes studied, transcripts for 652 host genes were differentially regulated between 5 and 16 hours-post-infection compared with only 25 between uninfected cells and 5 hours post-infection. NF-kB activated genes and lysosome components like S100 were upregulated, and chemokines such as CCL24, CXCL2, CXCL5 and CXCL8 downregulated.ImportanceAfrican swine fever virus (ASFV) causes haemorrhagic fever in domestic pigs with case fatality rates approaching 100%, and no approved vaccines or antivirals. The highly-virulent ASFV Georgia 2007/1 strain (GRG) was the first isolated when ASFV spread from Africa to the Caucasus region in 2007. Then spreading through Eastern Europe, and more recently across Asia. We used an RNA-based next generation sequencing technique called CAGE-seq to map the starts of viral genes across the GRG DNA genome. This has allowed us to investigate which viral genes are expressed during early or late stages of infection and how this is controlled, comparing their expression to the non-virulent ASFV-BA71V strain to identify key genes that play a role in virulence. In parallel we investigated how host cells respond to infection, which revealed how the ASFV suppresses components of the host immune response to ultimately win the arms race against its porcine host.

RNA polymerase inhibition plays an important role in the regulation of transcription in response to environmental changes and in the virus-host relationship. Here we present the high-resolution structures of two such RNAP-inhibitor complexes that provide the structural bases underlying RNAP inhibition in archaea. The Acidianus two-tailed virus encodes the RIP factor that binds inside the DNA-binding channel of RNAP, inhibiting transcription by occlusion of binding sites for nucleic acid and the transcription initiation factor TFB. Infection with the Sulfolobus Turreted Icosahedral Virus induces the expression of the host factor TFS4, which binds in the RNAP funnel similarly to eukaryotic transcript cleavage factors. However, TFS4 allosterically induces a widening of the DNA-binding channel which disrupts trigger loop and bridge helix motifs. Importantly, the conformational changes induced by TFS4 are closely related to inactivated states of RNAP in other domains of life indicating a deep evolutionary conservation of allosteric RNAP inhibition., Understanding the structural basis for the inhibition of archaeal eukaryotic-like RNA polymerases (RNAPs) during virus infection is of interest for drug design. Here, the authors present the cryo-EM structures of apo Sulfolobus acidocaldarius RNAP and the RNAP complex structures with two regulatory factors, RIP and TFS4 that inhibit transcription and discuss their inhibitory mechanisms.

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.

Recruitment of RNA polymerase and initiation factors to the promoter is the only known target for transcription activation and repression in archaea. Whether any of the subsequent steps towards productive transcription elongation are involved in regulation is not known. We characterised how the basal transcription machinery is distributed along genes in the archaeon Saccharolobus solfataricus. We discovered a distinct early elongation phase where RNA polymerases sequentially recruit the elongation factors Spt4/5 and Elf1 to form the transcription elongation complex (TEC) before the TEC escapes into productive transcription. TEC escape is rate-limiting for transcription output during exponential growth. Oxidative stress causes changes in TEC escape that correlate with changes in the transcriptome. Our results thus establish that TEC escape contributes to the basal promoter strength and facilitates transcription regulation. Impaired TEC escape coincides with the accumulation of initiation factors at the promoter and recruitment of termination factor aCPSF1 to the early TEC. This suggests two possible mechanisms for how TEC escape limits transcription, physically blocking upstream RNA polymerases during transcription initiation and premature termination of early TECs., Transcription in archaea is known to be regulated through the recruitment of RNA polymerase to promoters. Here, the authors show that the archaeon Saccharolobus solfataricus regulates transcription globally through a rate-limiting promoter-proximal elongation step.

Evolution-related multisubunit RNA polymerases (RNAPs) carry out RNA synthesis in all domains life. Although their catalytic cores and fundamental mechanisms of transcription elongation are conserved, the initiation stage of the transcription cycle differs substantially in bacteria, archaea, and eukaryotes in terms of the requirements for accessory factors and details of the molecular mechanisms. This review focuses on recent insights into the evolution of the transcription apparatus with regard to (a) the surprisingly pervasive double-Ψ β-barrel active-site configuration among different nucleic acid polymerase families, (b) the origin and phylogenetic distribution of TBP, TFB, and TFE transcription factors, and (c) the functional relationship between transcription and translation initiation mechanisms in terms of transcription start site selection and RNA structure.

Virus-encoded transcription factors have been pivotal in exploring the molecular mechanisms and regulation of gene expression in bacteria and eukaryotes since the birth of molecular biology, while our understanding of viral transcription in archaea is still in its infancy. Archaeal viruses do not encode their own RNA polymerases (RNAPs) and are consequently entirely dependent on their hosts for gene expression; this is fundamentally different from many bacteriophages and requires alternative regulatory strategies. Archaeal viruses wield a repertoire of proteins to expropriate the host transcription machinery to their own benefit. In this short review we summarise our current understanding of gene-specific and global mechanisms that viruses employ to chiefly downregulate host transcription and enable the efficient and temporal expression of the viral transcriptome. Most of the experimentally characterised archaeo-viral transcription regulators possess either ribbon-helix-helix or Zn-finger motifs that allow them to engage with the DNA in a sequence-specific manner, altering the expression of a specific subset of genes. Recently a novel type of regulator was reported that directly binds to the RNAP and shuts down transcription of both host and viral genes in a global fashion.

The African Swine Fever Virus causes haemorrhagic fever in domestic pigs and presents the biggest global threat to animal farming in recorded history. Despite its importance, very little is known the mechanisms and temporal regulation of transcription in ASFV. Here we report the first detailed viral transcriptome analysis of ASFV during early and late infection ofVerocells. In addition to total RNA sequencing, we have characterised the transcription start sites and transcription termination sites at nucleotide-resolution, revealing the distinct DNA consensus motifs of early and late promoters, as well as the sequence determinants for transcription termination. ASFV can utilise alternative promoters to generate distinct proteins from the same transcription unit that differ with respect to the polypeptide N-terminus. Finally, our results reveal that the ASFV-RNAP undergoes transcript slippage at the 5’ end of transcription units that in a promoter sequence-specific manner results in the addition of 5’-AT and 5’-ATAT tails to mRNAs.

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.

Prokaryotic Argonaute proteins acquire guide strands derived from invading or mobile genetic elements, via an unknown pathway, to direct guide-dependent cleavage of foreign DNA. Here, we report that Argonaute from the archaeal organism Methanocaldococcus jannaschii (MjAgo) possesses two modes of action: the canonical guide-dependent endonuclease activity and a non-guided DNA endonuclease activity. The latter allows MjAgo to process long double-stranded DNAs, including circular plasmid DNAs and genomic DNAs. Degradation of substrates in a guide-independent fashion primes MjAgo for subsequent rounds of DNA cleavage. Chromatinized genomic DNA is resistant to MjAgo degradation, and recombinant histones protect DNA from cleavage in vitro. Mutational analysis shows that key residues important for guide-dependent target processing are also involved in guide-independent MjAgo function. This is the first characterization of guide-independent cleavage activity for an Argonaute protein potentially serving as a guide biogenesis pathway in a prokaryotic system.

The archaeal transcription apparatus is closely related to the eukaryotic RNA polymerase (RNAP) II system, while\ud archaeal genomes are more similar to bacteria with densely packed genes organized in operons. This makes understanding\ud transcription in archaea vital, both in terms of molecular mechanisms and evolution. Very little is known about how\ud archaeal cells orchestrate transcription on a systems level. We have characterized the genome-wide occupancy of the\ud Methanocaldococcus jannaschii transcription machinery and its transcriptome. Our data reveal how the TATA and BRE\ud promoter elements facilitate recruitment of the essential initiation factors TATA-binding protein and transcription factor B,\ud respectively, which in turn are responsible for the loading of RNAP into the transcription units. The occupancies of\ud RNAP and Spt4/5 strongly correlate with each other and with RNA levels. Our results show that Spt4/5 is a general\ud elongation factor in archaea as its presence on all genes matches RNAP. Spt4/5 is recruited proximal to the transcription\ud start site on the majority of transcription units, while on a subset of genes, including rRNA and CRISPR loci, Spt4/5 is\ud recruited to the transcription elongation complex during early elongation within 500 base pairs of the transcription start\ud site and akin to its bacterial homologue NusG.

Archaea employ a eukaryote-like transcription apparatus to transcribe a bacteria-like genome; while the RNA polymerase, basal factors and promoter elements mirror the eukaryotic RNA polymerase II system, archaeal genomes are densely packed with genes organised into multicistronic transcription units. The molecular mechanisms of archaeal transcription have been studied and characterised in great detail in vitro, but until recently relatively little was known about its global characteristics. In this chapter we discuss an integrated view of transcription from the molecular to the global level. Systems biology approaches have provided compelling insights into promoter and terminator DNA elements, the genome-wide distribution of transcription initiation- and elongation factors and RNA polymerase, the archaeal transcriptome and chromatin organisation. Overall these analyses illuminate transcription from a genome-wide perspective and serve as a resource for the community. In addition, Big Data can often validate mechanistic models based on biochemical and structural information, and generate new working hypotheses that can be thoroughly tested and dissected in vitro. This is an exciting time to study gene expression in the archaea since we are at the brink of a comprehensive yet detailed understanding of transcription.

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.

The TATA binding protein (TBP) is an essential transcription initiation factor in Archaea and Eucarya. Bacteria lack TBP, and instead use sigma factors for transcription initiation. TBP has a symmetric structure comprising two repeated TBP domains. Using sequence, structural and phylogenetic analyses, we examine the distribution and evolutionary history of the TBP domain, a member of the helix-grip fold family. Our analyses reveal a broader distribution than for TBP, with TBP-domains being present across all three domains of life. In contrast to TBP, all other characterized examples of the TBP domain are present as single copies, primarily within multidomain proteins. The presence of the TBP domain in the ubiquitous DNA glycosylases suggests that this fold traces back to the ancestor of all three domains of life. The TBP domain is also found in RNase HIII, and phylogenetic analyses show that RNase HIII has evolved from bacterial RNase HII via TBP-domain fusion. Finally, our comparative genomic screens confirm and extend earlier reports of proteins consisting of a single TBP domain among some Archaea. These monopartite TBP-domain proteins suggest that this domain is functional in its own right, and that the TBP domain could have first evolved as an independent protein, which was later recruited in different contexts.

All RNAPs (RNA polymerases) repeatedly make use of their DNA template by progressing through the transcription cycle multiple times. During transcription initiation and elongation, distinct sets of transcription factors associate with multisubunit RNAPs and modulate their nucleic-acid-binding and catalytic properties. Between the initiation and elongation phases of the cycle, the factors have to be exchanged by a largely unknown mechanism. We have shown that the binding sites for initiation and elongation factors are overlapping and that the binding of the factors to RNAP is mutually exclusive. This ensures an efficient exchange or ‘swapping’ of factors and could furthermore assist RNAP during promoter escape, enabling robust transcription. A similar mechanism applies to the bacterial RNAP system. The elongation factors are evolutionarily conserved between the bacterial (NusG) and archaeo-eukaryotic (Spt5) systems; however, the initiation factors [σ and TBP (TATA-box-binding protein)/TF (transcription factor) B respectively] are not. Therefore we propose that this factor-swapping mechanism, operating in all three domains of life, is the outcome of convergent evolution.

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.

The molecular machines that carry out transcription in all domains of life—bacteria, archaea, and eukaryotes—are multisubunit RNA polymerases ( 1 ). Over the past 15 years, structural analyses at ever higher resolution, in particular hybrid approaches that combine x-ray crystallography and single-particle cryo–electron microscopy, have provided detailed insights into how these enzymes work ( 2 ). On page 921 of this issue, Ehara et al. ( 3 ) apply such a multidisciplinary structural approach and in vitro transcription assays to reveal functional insights into a complete elongation complex from the yeast Komagataella pastoris , encompassing RNA polymerase II (RNAPII) and the transcription elongation factors Elf1, Spt4/5, and TFIIS. The results uncover the detailed molecular mechanisms by which these factors can not only enhance transcription elongation, but also pausing.

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

RNA polymerases (RNAPs) carry out transcription in all living organisms. All multisubunit RNAPs are derived from a common ancestor, a fact that becomes apparent from their amino acid sequence, subunit composition, structure, function and molecular mechanisms. Despite the similarity of these complexes, the organisms that depend on them are extremely diverse, ranging from microorganisms to humans. Recent findings about the molecular and functional architecture of RNAPs has given us intriguing insights into their evolution and how their activities are harnessed by homologous and analogous basal factors during the transcription cycle. We provide an overview of the evolutionary conservation of and differences between the multisubunit polymerases in the three domains of life, and introduce the 'elongation first' hypothesis for the evolution of transcriptional regulation.

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.

Evolutionarily related multisubunit RNA polymerases (RNAPs) facilitate gene transcription throughout the three domains of life. During the past seven years an increasing number of bacterial and eukaryotic RNAP structures have been solved; however, the archaeal enzyme remained elusive. Two reports from the Murakami and Cramer laboratories have now filled this gap in our knowledge and enable us to hypothesize about the evolution of the structure and function of RNAPs.

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.

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.

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 archaeal genome is organized by either eukaryotic-like histone proteins or bacterial-like nucleoid-associated proteins. Recent studies have revealed novel insights into chromatin dynamics and their effect on gene expression in archaeal model organisms. In this Progress article, we discuss the interplay between chromatin proteins, such as histones and Alba, and components of the basal transcription machinery, as well as between chromatin structure and gene-specific transcription factors in archaea. Such an interplay suggests that chromatin might have a role in regulating gene expression on both a global and a gene-specific level. Moreover, several archaeal transcription factors combine a global gene regulatory role with an architectural role, thus contributing to chromatin organization and compaction, as well as gene expression. We describe the emerging principles underlying how these factors cooperate in nucleoid structuring and gene regulation.

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.

The urokinase-type plasminogen activator receptor (uPAR) is a glycolipid-anchored membrane protein that is thought to play an active role during cancer cell invasion and metastasis. We have expressed a truncated soluble form of human uPAR using its native signal peptide in stably transfected Drosophila Schneider 2 (S2) cells. This recombinant product, denoted suPAR (residues 1-283), is secreted in high quantities in serum-free medium and can be isolated in very high purity. Characterization by SDS-PAGE and mass spectrometry reveals that suPAR produced in this system carries a uniform glycosylation composed of biantennary carbohydrates. In contrast, suPAR produced in stably transfected Chinese hamster ovary (CHO) cells carries predominantly complex-type glycosylation and exhibits in addition a site-specific microheterogeneity of the individual N-linked carbohydrates. Measurement of binding kinetics for the interaction with uPA by surface plasmon resonance reveals that S2-produced suPAR exhibits binding properties similar to those of suPAR produced by CHO cells. By site-directed mutagenesis we have furthermore removed the five potential N-linked glycosylation-sites either individually or in various combinations and studied the effect thereof on secretion and ligand-binding. Only suPAR completely deprived of N-linked glycosylation exhibits an impaired level of secretion. All the other mutants showed comparable secretion levels and retained the ligand-binding properties of suPAR-wt. In conclusion, stable expression of suPAR in Drosophila S2 cells offers a convenient and attractive method for the large scale production of homogeneous preparations of several uPAR mutants, which may be required for future attempts to solve the three-dimensional structure of uPAR by X-ray crystallography.

Using a combination of genetic, biochemical, and structural approaches, we show that the cyclic-peptide antibiotic GE23077 (GE) binds directly to the bacterial RNA polymerase (RNAP) active-center 'i' and 'i+1' nucleotide binding sites, preventing the binding of initiating nucleotides, and thereby preventing transcription initiation. The target-based resistance spectrum for GE is unusually small, reflecting the fact that the GE binding site on RNAP includes residues of the RNAP active center that cannot be substituted without loss of RNAP activity. The GE binding site on RNAP is different from the rifamycin binding site. Accordingly, GE and rifamycins do not exhibit cross-resistance, and GE and a rifamycin can bind simultaneously to RNAP. The GE binding site on RNAP is immediately adjacent to the rifamycin binding site. Accordingly, covalent linkage of GE to a rifamycin provides a bipartite inhibitor having very high potency and very low susceptibility to target-based resistance. DOI: http://dx.doi.org/10.7554/eLife.02450.001.

The archaeal and eukaryotic evolutionary domains diverged from each other approximately 2 billion years ago, but many of the core components of their transcriptional and translational machineries still display a readily recognizable degree of similarity in their primary structures. The F and P subunits present in archaeal RNA polymerases were only recently identified in a purified archaeal RNA polymerase preparation and, on the basis of localized sequence homologies, tentatively identified as archaeal versions of the eukaryotic RPB4 and RPB12 RNA polymerase subunits, respectively. We prepared recombinant versions of the F and P subunits from Methanococcus jannaschii and used them in in vitro and in vivo protein interaction assays to demonstrate that they interact with other archaeal subunits in a manner predicted from their eukaryotic counterparts. The overall structural conservation of the M. jannaschii F subunit, although not readily recognizable on the primary amino acid sequence level, is sufficiently high to allow the formation of an archaeal-human F-RPB7 hybrid complex.

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.

TFIIB-like general transcription factors are required for transcription initiation by all eukaryotic and archaeal RNA polymerases (RNAPs). TFIIB facilitates both recruitment and post-recruitment steps of initiation; in particular, TFIIB stimulates abortive initiation. X-ray crystallography of TFIIB-RNAP II complexes shows that the TFIIB linker region penetrates the RNAP active center, yet the impact of this arrangement on RNAP activity and underlying mechanisms remains elusive. Promoter-independent abortive initiation assays exploit the intrinsic ability of RNAP enzymes to initiate transcription from nicked DNA templates and record the formation of the first phosphodiester bonds. These assays can be used to measure the effect of transcription factors such as TFIIB and RNAP mutations on abortive transcription.

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.

Site specific incorporation of molecular probes such as fluorescent- and nitroxide spin-labels into biomolecules, and subsequent analysis by Forster resonance energy transfer (FRET) and double electron-electron resonance (DEER) can elucidate the distance and distance-changes between the probes. However, the probes have an intrinsic conformational flexibility due to the linker by which they are conjugated to the biomolecule. This property minimizes the influence of the label side chain on the structure of the target molecule, but complicates the direct correlation of the experimental inter-label distances with the macromolecular structure or changes thereof. Simulation methods that account for the conformational flexibility and orientation of the probe(s) can be helpful in overcoming this problem. We performed distance measurements using FRET and DEER and explored different simulation techniques to predict inter-label distances using the Rpo4/7 stalk module of the M. jannaschii RNA polymerase. This is a suitable model system because it is rigid and a high-resolution X-ray structure is available. The conformations of the fluorescent labels and nitroxide spin labels on Rpo4/7 were modeled using in vacuo molecular dynamics simulations (MD) and a stochastic Monte Carlo sampling approach. For the nitroxide probes we also performed MD simulations with explicit water and carried out a rotamer library analysis. Our results show that the Monte Carlo simulations are in better agreement with experiments than the MD simulations and the rotamer library approach results in plausible distance predictions. Because the latter is the least computationally demanding of the methods we have explored, and is readily available to many researchers, it prevails as the method of choice for the interpretation of DEER distance distributions.

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.

The complex organization of the transcription machinery has been revealed mainly by biochemical and crystallographic studies. X-ray structures describe RNA polymerases and transcription complexes on an atomic level, but fail to portray their dynamic nature. The use of fluorescence techniques has made it possible to add a new layer of information to our understanding of transcription by providing details about the structural rearrangement of mobile elements and the network of interactions within transcription complexes in solution and in real-time.

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.

All cellular life depends on multisubunit RNAPs (RNA polymerases) that are evolutionarily related through the three domains of life. Archaeal RNAPs encompass 12 subunits that contribute in different ways to the assembly and stability of the enzyme, nucleic acid binding, catalysis and specific regulatory interactions with transcription factors. The recent development of methods to reconstitute archaeal RNAP from recombinant materials in conjunction with structural information of multisubunit RNAPs present a potent opportunity to investigate the molecular mechanisms of transcription.

The archaeal basal transcriptional machinery consists of TBP (TATA-binding protein), TFB (transcription factor B; a homologue of eukaryotic TFIIB) and an RNA polymerase that is structurally very similar to eukaryotic RNA polymerase II. This constellation of factors is sufficient to assemble specifically on a TATA box-containing promoter and to initiate transcription at a specific start site. We have used this system to study the functional interaction between basal transcription factors and RNA polymerase, with special emphasis on the post-recruitment function of TFB. A bioinformatics analysis of the B-finger of archaeal TFB and eukaryotic TFIIB reveals that this structure undergoes rapid and apparently systematic evolution in archaeal and eukaryotic evolutionary domains. We provide a detailed analysis of these changes and discuss their possible functional implications.

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.

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.