Chromatin and gene regulation in archaea.

The chromatinisation of DNA by nucleoid-associated proteins (NAPs) in archaea 'formats' the genome structure in profound ways, revealing both striking differences and analogies to eukaryotic chromatin. However, the extent to which archaeal NAPs actively regulate gene expression remains poorly understood. The dawn of quantitative chromatin mapping techniques and first NAP-specific occupancy profiles in different archaea promise a more accurate view. A picture emerges where in diverse archaea with very different NAP repertoires chromatin maintains access to regulatory motifs including the gene promoter independently of transcription activity. Our re-analysis of genome-wide occupancy data of the crenarchaeal NAP Cren7 shows that these chromatin-free regions are flanked by increased Cren7 binding across the transcription start site. While bacterial NAPs often form heterochromatin-like regions across islands with xenogeneic genes that are transcriptionally silenced, there is little evidence for similar structures in archaea and data from Haloferax show that the promoters of xenogeneic genes remain accessible. Local changes in chromatinisation causing wide-ranging effects on transcription restricted to one chromosomal interaction domain (CID) in Saccharolobus islandicus hint at a higher-order level of organisation between chromatin and transcription. The emerging challenge is to integrate results obtained at microscale and macroscale, reconciling molecular structure and function with dynamic genome-wide chromatin landscapes.

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

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.

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.

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.

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.

Archaeology of RNA polymerase: factor swapping during the transcription cycle.

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.

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.

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.

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.

An HflX-type GTPase from Sulfolobus solfataricus binds to the 50S ribosomal subunit in all nucleotide-bound states.

HflX GTPases are found in all three domains of life, the Bacteria, Archaea, and Eukarya. HflX from Escherichia coli has been shown to bind to the 50S ribosomal subunit in a nucleotide-dependent manner, and this interaction strongly stimulates its GTPase activity. We recently determined the structure of an HflX ortholog from the archaeon Sulfolobus solfataricus (SsoHflX). It revealed the presence of a novel HflX domain that might function in RNA binding and is linked to a canonical G domain. This domain arrangement is common to all archaeal, bacterial, and eukaryotic HflX GTPases. This paper shows that the archaeal SsoHflX, like its bacterial orthologs, binds to the 50S ribosomal subunit. This interaction does not depend on the presence of guanine nucleotides. The HflX domain is sufficient for ribosome interaction. Binding appears to be restricted to free 50S ribosomal subunits and does not occur with 70S ribosomes engaged in translation. The fingerprint (1)H-(15)N heteronuclear correlation nuclear magnetic resonance (NMR) spectrum of SsoHflX reveals a large number of well-resolved resonances that are broadened upon binding to the 50S ribosomal subunit. The GTPase activity of SsoHflX is stimulated by crude fractions of 50S ribosomal subunits, but this effect is lost with further high-salt purification of the 50S ribosomal subunits, suggesting that the stimulation depends on an extrinsic factor bound to the 50S ribosomal subunit. Our results reveal common properties but also marked differences between archaeal and bacterial HflX proteins.

Assembling the archaeal ribosome: roles for translation-factor-related GTPases.

The assembly of ribosomal subunits from their individual components (rRNA and ribosomal proteins) requires the assistance of a multitude of factors in order to control and increase the efficiency of the assembly process. GTPases of the TRAFAC (translation-factor-related) class constitute a major type of ribosome-assembly factor in Eukaryota and Bacteria. They are thought to aid the stepwise assembly of ribosomal subunits through a 'molecular switch' mechanism that involves conformational changes in response to GTP hydrolysis. Most conserved TRAFAC GTPases are involved in ribosome assembly or other translation-associated processes. They typically interact with ribosomal subunits, but in many cases, the exact role that these GTPases play remains unclear. Previous studies almost exclusively focused on the systems of Bacteria and Eukaryota. Archaea possess several conserved TRAFAC GTPases as well, with some GTPase families being present only in the archaeo-eukaryotic lineage. In the present paper, we review the occurrence of TRAFAC GTPases with translation-associated functions in Archaea.

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.

Structure of the ribosome associating GTPase HflX.

The HflX-family is a widely distributed but poorly characterized family of translation factor-related guanosine triphosphatases (GTPases) that interact with the large ribosomal subunit. This study describes the crystal structure of HflX from Sulfolobus solfataricus solved to 2.0-A resolution in apo- and GDP-bound forms. The enzyme displays a two-domain architecture with a novel "HflX domain" at the N-terminus, and a classical G-domain at the C-terminus. The HflX domain is composed of a four-stranded parallel beta-sheet flanked by two alpha-helices on either side, and an anti-parallel coiled coil of two long alpha-helices that lead to the G-domain. The cleft between the two domains accommodates the nucleotide binding site as well as the switch II region, which mediates interactions between the two domains. Conformational changes of the switch regions are therefore anticipated to reposition the HflX-domain upon GTP-binding. Slow GTPase activity has been confirmed, with an HflX domain deletion mutant exhibiting a 24-fold enhanced turnover rate, suggesting a regulatory role for the HflX domain. The conserved positively charged surface patches of the HflX-domain may mediate interaction with the large ribosomal subunit. The present study provides a structural basis to uncover the functional role of this GTPases family whose function is largely unknown.

Fidelity in archaeal information processing.

A key element during the flow of genetic information in living systems is fidelity. The accuracy of DNA replication influences the genome size as well as the rate of genome evolution. The large amount of energy invested in gene expression implies that fidelity plays a major role in fitness. On the other hand, an increase in fidelity generally coincides with a decrease in velocity. Hence, an important determinant of the evolution of life has been the establishment of a delicate balance between fidelity and variability. This paper reviews the current knowledge on quality control in archaeal information processing. While the majority of these processes are homologous in Archaea, Bacteria, and Eukaryotes, examples are provided of nonorthologous factors and processes operating in the archaeal domain. In some instances, evidence for the existence of certain fidelity mechanisms has been provided, but the factors involved still remain to be identified.

Role of multiprotein bridging factor 1 in archaea: bridging the domains?

MBF1 (multiprotein bridging factor 1) is a highly conserved protein in archaea and eukaryotes. It was originally identified as a mediator of the eukaryotic transcription regulator BmFTZ-F1 (Bombyx mori regulator of fushi tarazu). MBF1 was demonstrated to enhance transcription by forming a bridge between distinct regulatory DNA-binding proteins and the TATA-box-binding protein. MBF1 consists of two parts: a C-terminal part that contains a highly conserved helix-turn-helix, and an N-terminal part that shows a clear divergence: in eukaryotes, it is a weakly conserved flexible domain, whereas, in archaea, it is a conserved zinc-ribbon domain. Although its function in archaea remains elusive, its function as a transcriptional co-activator has been deduced from thorough studies of several eukaryotic proteins, often indicating a role in stress response. In addition, MBF1 was found to influence translation fidelity in yeast. Genome context analysis of mbf1 in archaea revealed conserved clustering in the crenarchaeal branch together with genes generally involved in gene expression. It points to a role of MBF1 in transcription and/or translation. Experimental data are required to allow comparison of the archaeal MBF1 with its eukaryotic counterpart.

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.

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.

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.

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.