Cbp1 and Cren7 form chromatin-like structures that ensure efficient transcription of long CRISPR arrays.

CRISPR arrays form the physical memory of CRISPR adaptive immune systems by incorporating foreign DNA as spacers that are often AT-rich and derived from viruses. As promoter elements such as the TATA-box are AT-rich, CRISPR arrays are prone to harbouring cryptic promoters. Sulfolobales harbour extremely long CRISPR arrays spanning several kilobases, a feature that is accompanied by the CRISPR-specific transcription factor Cbp1. Aberrant Cbp1 expression modulates CRISPR array transcription, but the molecular mechanisms underlying this regulation are unknown. Here, we characterise the genome-wide Cbp1 binding at nucleotide resolution and characterise the binding motifs on distinct CRISPR arrays, as well as on unexpected non-canonical binding sites associated with transposons. Cbp1 recruits Cren7 forming together 'chimeric' chromatin-like structures at CRISPR arrays. We dissect Cbp1 function in vitro and in vivo and show that the third helix-turn-helix domain is responsible for Cren7 recruitment, and that Cbp1-Cren7 chromatinization plays a dual role in the transcription of CRISPR arrays. It suppresses spurious transcription from cryptic promoters within CRISPR arrays but enhances CRISPR RNA transcription directed from their cognate promoters in their leader region. Our results show that Cbp1-Cren7 chromatinization drives the productive expression of long CRISPR arrays.

DNA-bridging by an archaeal histone variant via a unique tetramerisation interface.

In eukaryotes, histone paralogues form obligate heterodimers such as H3/H4 and H2A/H2B that assemble into octameric nucleosome particles. Archaeal histones are dimeric and assemble on DNA into 'hypernucleosome' particles of varying sizes with each dimer wrapping 30 bp of DNA. These are composed of canonical and variant histone paralogues, but the function of these variants is poorly understood. Here, we characterise the structure and function of the histone paralogue MJ1647 from Methanocaldococcus jannaschii that has a unique C-terminal extension enabling homotetramerisation. The 1.9 Å X-ray structure of a dimeric MJ1647 species, structural modelling of the tetramer, and site-directed mutagenesis reveal that the C-terminal tetramerization module consists of two alpha helices in a handshake arrangement. Unlike canonical histones, MJ1647 tetramers can bridge two DNA molecules in vitro. Using single-molecule tethered particle motion and DNA binding assays, we show that MJ1647 tetramers bind ~60 bp DNA and compact DNA in a highly cooperative manner. We furthermore show that MJ1647 effectively competes with the transcription machinery to block access to the promoter in vitro. To the best of our knowledge, MJ1647 is the first histone shown to have DNA bridging properties, which has important implications for genome structure and gene expression in archaea.

A novel metagenome-derived viral RNA polymerase and its application in a cell-free expression system for metagenome screening.

The mining of genomes from non-cultivated microorganisms using metagenomics is a powerful tool to discover novel proteins and other valuable biomolecules. However, function-based metagenome searches are often limited by the time-consuming expression of the active proteins in various heterologous host systems. We here report the initial characterization of novel single-subunit bacteriophage RNA polymerase, EM1 RNAP, identified from a metagenome data set obtained from an elephant dung microbiome. EM1 RNAP and its promoter sequence are distantly related to T7 RNA polymerase. Using EM1 RNAP and a translation-competent Escherichia coli extract, we have developed an efficient medium-throughput pipeline and protocol allowing the expression of metagenome-derived genes and the production of proteins in cell-free system is sufficient for the initial testing of the predicted activities. Here, we have successfully identified and verified 12 enzymes acting on bis(2-hydroxyethyl) terephthalate (BHET) in a completely clone-free approach and proposed an in vitro high-throughput metagenomic screening method.

ChIP-Seq Occupancy Mapping of the Archaeal Transcription Machinery.

Genome-wide occupancy studies for RNA polymerases and their basal transcription factors deliver information about transcription dynamics and the recruitment of transcription elongation and termination factors in eukaryotes and prokaryotes. The primary method to determine genome-wide occupancies is chromatin immunoprecipitation combined with deep sequencing (ChIP-seq). Archaea possess a transcription machinery that is evolutionarily closer related to its eukaryotic counterpart but it operates in a prokaryotic cellular context. Studies on archaeal transcription brought insight into the evolution of transcription machineries and the universality of transcription mechanisms. Because of the limited resolution of ChIP-seq, the close spacing of promoters and transcription units found in archaeal genomes pose a challenge for ChIP-seq and the ensuing data analysis. The extreme growth temperature of many established archaeal model organisms necessitates further adaptations. This chapter describes a version of ChIP-seq adapted for the basal transcription machinery of thermophilic archaea and some modifications to the data analysis.

Promoter-proximal elongation regulates transcription in archaea.

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.

Key Concepts and Challenges in Archaeal Transcription.

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.

Structural and functional adaptation of Haloferax volcanii TFEα/ß.

The basal transcription factor TFE enhances transcription initiation by catalysing DNA strand-separation, a process that varies with temperature and ionic strength. Canonical TFE forms a heterodimeric complex whose integrity and function critically relies on a cubane iron-sulphur cluster residing in the TFEß subunit. Halophilic archaea such as Haloferax volcanii have highly divergent putative TFEß homologues with unknown properties. Here, we demonstrate that Haloferax TFEß lacks the prototypical iron-sulphur cluster yet still forms a stable complex with TFEα. A second metal cluster contained in the zinc ribbon domain in TFEα is highly degenerate but retains low binding affinity for zinc, which contributes to protein folding and stability. The deletion of the tfeB gene in H. volcanii results in the aberrant expression of approximately one third of all genes, consistent with its function as a basal transcription initiation factor. Interestingly, tfeB deletion particularly affects foreign genes including a prophage region. Our results reveal the loss of metal centres in Hvo transcription factors, and confirm the dual function of TFE as basal factor and regulator of transcription.

The cutting edge of archaeal transcription.

The archaeal RNA polymerase (RNAP) is a double-psi ß-barrel enzyme 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. Understanding archaeal transcription is, therefore, key to delineate the universally conserved fundamental mechanisms of transcription as well as the evolution of the archaeo-eukaryotic transcription machineries. The dynamic interplay between RNAP subunits, transcription factors and nucleic acids dictates the activity of RNAP and ultimately gene expression. This review focusses on recent progress in our understanding of (i) the structure, function and molecular mechanisms of known and less characterized factors including Elf1 (Elongation factor 1), NusA (N-utilization substance A), TFS4, RIP and Eta, and (ii) their evolution and phylogenetic distribution across the expanding tree of Archaea.

The transcript cleavage factor paralogue TFS4 is a potent RNA polymerase inhibitor.

TFIIS-like transcript cleavage factors enhance the processivity and fidelity of archaeal and eukaryotic RNA polymerases. Sulfolobus solfataricus TFS1 functions as a bona fide cleavage factor, while the paralogous TFS4 evolved into a potent RNA polymerase inhibitor. TFS4 destabilises the TBP-TFB-RNAP pre-initiation complex and inhibits transcription initiation and elongation. All inhibitory activities are dependent on three lysine residues at the tip of the C-terminal zinc ribbon of TFS4; the inhibition likely involves an allosteric component and is mitigated by the basal transcription factor TFEα/ß. A chimeric variant of yeast TFIIS and TFS4 inhibits RNAPII transcription, suggesting that the molecular basis of inhibition is conserved between archaea and eukaryotes. TFS4 expression in S. solfataricus is induced in response to infection with the S ulfolobus turreted icosahedral virus. Our results reveal a compelling functional diversification of cleavage factors in archaea, and provide novel insights into transcription inhibition in the context of the host-virus relationship.

Evolutionary Origins of Two-Barrel RNA Polymerases and Site-Specific Transcription Initiation.

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 ß-barrel active-site configuration among different nucleic acid polymerase families, (b) the origin and phylogenetic distribution of TBP, TFB, and TFE transcription factors, and

A global analysis of transcription reveals two modes of Spt4/5 recruitment to archaeal RNA polymerase.

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

Same same but different: The evolution of TBP in archaea and their eukaryotic offspring.

Transcription factors TBP and TF(II)B assemble with RNA polymerase at the promoter DNA forming the initiation complex. Despite a high degree of conservation, the molecular binding mechanisms of archaeal and eukaryotic TBP and TF(II)B differ significantly. Based on recent biophysical data, we speculate how the mechanisms co-evolved with transcription regulation and TBP multiplicity.

Repression of RNA polymerase by the archaeo-viral regulator ORF145/RIP.

Little is known about how archaeal viruses perturb the transcription machinery of their hosts. Here we provide the first example of an archaeo-viral transcription factor that directly targets the host RNA polymerase (RNAP) and efficiently represses its activity. ORF145 from the temperate Acidianus two-tailed virus (ATV) forms a high-affinity complex with RNAP by binding inside the DNA-binding channel where it locks the flexible RNAP clamp in one position. This counteracts the formation of transcription pre-initiation complexes in vitro and represses abortive and productive transcription initiation, as well as elongation. Both host and viral promoters are subjected to ORF145 repression. Thus, ORF145 has the properties of a global transcription repressor and its overexpression is toxic for Sulfolobus. On the basis of its properties, we have re-named ORF145 RNAP Inhibitory Protein (RIP).

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

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 ß-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.

Archaeal TFEα/ß is a hybrid of TFIIE and the RNA polymerase III subcomplex hRPC62/39.

Transcription initiation of archaeal RNA polymerase (RNAP) and eukaryotic RNAPII is assisted by conserved basal transcription factors. The eukaryotic transcription factor TFIIE consists of α and ß subunits. Here we have identified and characterised the function of the TFIIEß homologue in archaea that on the primary sequence level is related to the RNAPIII subunit hRPC39. Both archaeal TFEß and hRPC39 harbour a cubane 4Fe-4S cluster, which is crucial for heterodimerization of TFEα/ß and its engagement with the RNAP clamp. TFEα/ß stabilises the preinitiation complex, enhances DNA melting, and stimulates abortive and productive transcription. These activities are strictly dependent on the ß subunit and the promoter sequence. Our results suggest that archaeal TFEα/ß is likely to represent the evolutionary ancestor of TFIIE-like factors in extant eukaryotes.

Transcription in Archaea: preparation of Methanocaldococcus jannaschii transcription machinery.

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.

Transcription in Archaea: in vitro transcription assays for mjRNAP.

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 MBF1 binds to 30S and 70S ribosomes via its helix-turn-helix domain.

MBF1 (multi-protein bridging factor 1) is a protein containing a conserved HTH (helix-turn-helix) domain in both eukaryotes and archaea. Eukaryotic MBF1 has been reported to function as a transcriptional co-activator that physically bridges transcription regulators with the core transcription initiation machinery of RNA polymerase II. In addition, MBF1 has been found to be associated with polyadenylated mRNA in yeast as well as in mammalian cells. aMBF1 (archaeal MBF1) is very well conserved among most archaeal lineages; however, its function has so far remained elusive. To address this, we have conducted a molecular characterization of this aMBF1. Affinity purification of interacting proteins indicates that aMBF1 binds to ribosomal subunits. On sucrose density gradients, aMBF1 co-fractionates with free 30S ribosomal subunits as well as with 70S ribosomes engaged in translation. Binding of aMBF1 to ribosomes does not inhibit translation. Using NMR spectroscopy, we show that aMBF1 contains a long intrinsically disordered linker connecting the predicted N-terminal zinc-ribbon domain with the C-terminal HTH domain. The HTH domain, which is conserved in all archaeal and eukaryotic MBF1 homologues, is directly involved in the association of aMBF1 with ribosomes. The disordered linker of the ribosome-bound aMBF1 provides the N-terminal domain with high flexibility in the aMBF1-ribosome complex. Overall, our findings suggest a role for aMBF1 in the archaeal translation process.

Eukaryotic and archaeal TBP and TFB/TF(II)B follow different promoter DNA bending pathways.

During transcription initiation, the promoter DNA is recognized and bent by the basal transcription factor TATA-binding protein (TBP). Subsequent association of transcription factor B (TFB) with the TBP-DNA complex is followed by the recruitment of the ribonucleic acid polymerase resulting in the formation of the pre-initiation complex. TBP and TFB/TF(II)B are highly conserved in structure and function among the eukaryotic-archaeal domain but intriguingly have to operate under vastly different conditions. Employing single-pair fluorescence resonance energy transfer, we monitored DNA bending by eukaryotic and archaeal TBPs in the absence and presence of TFB in real-time. We observed that the lifetime of the TBP-DNA interaction differs significantly between the archaeal and eukaryotic system. We show that the eukaryotic DNA-TBP interaction is characterized by a linear, stepwise bending mechanism with an intermediate state distinguished by a distinct bending angle. TF(II)B specifically stabilizes the fully bent TBP-promoter DNA complex and we identify this step as a regulatory checkpoint. In contrast, the archaeal TBP-DNA interaction is extremely dynamic and TBP from the archaeal organism Sulfolobus acidocaldarius strictly requires TFB for DNA bending. Thus, we demonstrate that transcription initiation follows diverse pathways on the way to the formation of the pre-initiation complex.

Differential translation tunes uneven production of operon-encoded proteins.

Clustering of functionally related genes in operons allows for coregulated gene expression in prokaryotes. This is advantageous when equal amounts of gene products are required. Production of protein complexes with an uneven stoichiometry, however, requires tuning mechanisms to generate subunits in appropriate relative quantities. Using comparative genomic analysis, we show that differential translation is a key determinant of modulated expression of genes clustered in operons and that codon bias generally is the best in silico indicator of unequal protein production. Variable ribosome density profiles of polycistronic transcripts correlate strongly with differential translation patterns. In addition, we provide experimental evidence that de novo initiation of translation can occur at intercistronic sites, allowing for differential translation of any gene irrespective of its position on a polycistronic messenger. Thus, modulation of translation efficiency appears to be a universal mode of control in bacteria and archaea that allows for differential production of operon-encoded proteins.