Structural basis of archaeal RNA polymerase transcription elongation and Spt4/5 recruitment.

Archaeal transcription is carried out by a multi-subunit RNA polymerase (RNAP) that is highly homologous in structure and function to eukaryotic RNAP II. Among the set of basal transcription factors, only Spt5 is found in all domains of life, but Spt5 has been shaped during evolution, which is also reflected in the heterodimerization of Spt5 with Spt4 in Archaea and Eukaryotes. To unravel the mechanistic basis of Spt4/5 function in Archaea, we performed structure-function analyses using the archaeal transcriptional machinery of Pyrococcus furiosus (Pfu). We report single-particle cryo-electron microscopy reconstructions of apo RNAP and the archaeal elongation complex (EC) in the absence and presence of Spt4/5. Surprisingly, Pfu Spt4/5 also binds the RNAP in the absence of nucleic acids in a distinct super-contracted conformation. We show that the RNAP clamp/stalk module exhibits conformational flexibility in the apo state of RNAP and that the enzyme contracts upon EC formation or Spt4/5 engagement. We furthermore identified a contact of the Spt5-NGN domain with the DNA duplex that stabilizes the upstream boundary of the transcription bubble and impacts Spt4/5 activity in vitro. This study, therefore, provides the structural basis for Spt4/5 function in archaeal transcription and reveals a potential role beyond the well-described support of elongation.

Uncovering the temporal dynamics and regulatory networks of thermal stress response in a hyperthermophile using transcriptomics and proteomics.

Facing rapid fluctuations in their natural environment, extremophiles, like the hyperthermophilic archaeon Pyrococcus furiosus, exhibit remarkable adaptability to extreme conditions. However, our understanding of their dynamic cellular responses remains limited. This study integrates RNA-sequencing and mass spectrometry data, thereby elucidating transcriptomic and proteomic responses to heat and cold shock stress in P. furiosus. Our results reveal rapid and dynamic changes in gene and protein expression following these stress responses. Heat shock triggers extensive transcriptome reprogramming, orchestrated by the transcriptional regulator Phr, targeting a broader gene repertoire than previously demonstrated. For heat shock signature genes, RNA levels swiftly return to baseline upon recovery, while protein levels remain persistently upregulated, reflecting a rapid but sustained response. Intriguingly, cold shock at 4°C elicits distinct short- and long-term responses at both RNA and protein levels. Cluster analysis identified gene sets with either congruent or contrasting trends in RNA and protein changes, representing well-separated arCOG groups tailored to their individual cellular responses. Particularly, upregulation of ribosomal proteins and significant enrichment of 5'-leadered sequences in cold-shock responsive genes suggest that translation regulation is important during cold shock adaption. Further investigating transcriptomic features, we reveal that thermal stress genes are equipped with basal sequence elements, such as strong promoter and poly(U)-terminators, facilitating a regulated response of the respective transcription units. Our study provides a comprehensive overview of the cellular response to temperature stress, advancing our understanding of stress response mechanisms in hyperthermophilic archaea and providing valuable insights into the molecular adaptations that facilitate life in extreme environments.IMPORTANCEExtreme environments provide unique challenges for life, and the study of extremophiles can shed light on the mechanisms of adaptation to such conditions. Pyrococcus furiosus, a hyperthermophilic archaeon, is a model organism for studying thermal stress response mechanisms. In this study, we used an integrated analysis of RNA-sequencing and mass spectrometry data to investigate the transcriptomic and proteomic responses of P. furiosus to heat and cold shock stress and recovery. Our results reveal the rapid and dynamic changes in gene and protein expression patterns associated with these stress responses, as well as the coordinated regulation of different gene sets in response to different stressors. These findings provide valuable insights into the molecular adaptations that facilitate life in extreme environments and advance our understanding of stress response mechanisms in hyperthermophilic archaea.

The archaeal Lsm protein from Pyrococcus furiosus binds co-transcriptionally to poly(U)-rich target RNAs.

Posttranscriptional processes in Bacteria include the association of small regulatory RNAs (sRNA) with a target mRNA. The sRNA/mRNA annealing process is often mediated by an RNA chaperone called Hfq. The functional role of bacterial and eukaryotic Lsm proteins is partially understood, whereas knowledge about archaeal Lsm proteins is scarce. Here, we used the genetically tractable archaeal hyperthermophile Pyrococcus furiosus to identify the protein interaction partners of the archaeal Sm-like proteins (PfuSmAP1) using mass spectrometry and performed a transcriptome-wide binding site analysis of PfuSmAP1. Most of the protein interaction partners we found are part of the RNA homoeostasis network in Archaea including ribosomal proteins, the exosome, RNA-modifying enzymes, but also RNA polymerase subunits, and transcription factors. We show that PfuSmAP1 preferentially binds messenger RNAs and antisense RNAs recognizing a gapped poly(U) sequence with high affinity. Furthermore, we found that SmAP1 co-transcriptionally associates with target RNAs. Our study reveals that in contrast to bacterial Hfq, PfuSmAP1 does not affect the transcriptional activity or the pausing behaviour of archaeal RNA polymerases. We propose that PfuSmAP1 recruits antisense RNAs to target mRNAs and thereby executes its putative regulatory function on the posttranscriptional level.

Insights into synthesis and function of KsgA/Dim1-dependent rRNA modifications in archaea.

Ribosomes are intricate molecular machines ensuring proper protein synthesis in every cell. Ribosome biogenesis is a complex process which has been intensively analyzed in bacteria and eukaryotes. In contrast, our understanding of the in vivo archaeal ribosome biogenesis pathway remains less characterized. Here, we have analyzed the in vivo role of the almost universally conserved ribosomal RNA dimethyltransferase KsgA/Dim1 homolog in archaea. Our study reveals that KsgA/Dim1-dependent 16S rRNA dimethylation is dispensable for the cellular growth of phylogenetically distant archaea. However, proteomics and functional analyses suggest that archaeal KsgA/Dim1 and its rRNA modification activity (i) influence the expression of a subset of proteins and (ii) contribute to archaeal cellular fitness and adaptation. In addition, our study reveals an unexpected KsgA/Dim1-dependent variability of rRNA modifications within the archaeal phylum. Combining structure-based functional studies across evolutionary divergent organisms, we provide evidence on how rRNA structure sequence variability (re-)shapes the KsgA/Dim1-dependent rRNA modification status. Finally, our results suggest an uncoupling between the KsgA/Dim1-dependent rRNA modification completion and its release from the nascent small ribosomal subunit. Collectively, our study provides additional understandings into principles of molecular functional adaptation, and further evolutionary and mechanistic insights into an almost universally conserved step of ribosome synthesis.

CopR, a Global Regulator of Transcription to Maintain Copper Homeostasis in Pyrococcus furiosus.

Although copper is in many cases an essential micronutrient for cellular life, higher concentrations are toxic. Therefore, all living cells have developed strategies to maintain copper homeostasis. In this manuscript, we have analyzed the transcriptome-wide response of Pyrococcus furiosus to increased copper concentrations and described the essential role of the putative copper-sensing metalloregulator CopR in the detoxification process. To this end, we employed biochemical and biophysical methods to characterize the role of CopR. Additionally, a copR knockout strain revealed an amplified sensitivity in comparison to the parental strain towards increased copper levels, which designates an essential role of CopR for copper homeostasis. To learn more about the CopR-regulated gene network, we performed differential gene expression and ChIP-seq analysis under normal and 20 µM copper-shock conditions. By integrating the transcriptome and genome-wide binding data, we found that CopR binds to the upstream regions of many copper-induced genes. Negative-stain transmission electron microscopy and 2D class averaging revealed an octameric assembly formed from a tetramer of dimers for CopR, similar to published crystal structures from the Lrp family. In conclusion, we propose a model for CopR-regulated transcription and highlight the regulatory network that enables Pyrococcus to respond to increased copper concentrations.

Next Generation DNA-Seq and Differential RNA-Seq Allow Re-annotation of the Pyrococcus furiosus DSM 3638 Genome and Provide Insights Into Archaeal Antisense Transcription.

Pyrococcus furiosus DSM 3638 is a model organism for hyperthermophilic archaea with an optimal growth temperature near 100°C. The genome was sequenced about 18 years ago. However, some publications suggest that in contrast to other Pyrococcus species, the genome of P. furiosus DSM 3638 is prone to genomic rearrangements. Therefore, we re-sequenced the genome using third generation sequencing techniques. The new de novo assembled genome is 1,889,914 bp in size and exhibits high sequence identity to the published sequence. However, two major deviations were detected: (1) The genome is 18,342 bp smaller than the NCBI reference genome due to a recently described deletion. (2) The region between PF0349 and PF0388 is inverted most likely due an assembly problem for the original sequence. In addition, numerous minor variations, ranging from single nucleotide exchanges, deletions or insertions were identified. The total number of insertion sequence (IS) elements is also reduced from 30 to 24 in the new sequence. Re-sequencing of a 2-year-old "lab culture" using Nanopore sequencing confirmed the overall stability of the P. furiosus DSM 3638 genome even under normal lab conditions without taking any special care. To improve genome annotation, the updated DNA sequence was combined with an RNA sequencing approach. Here, RNAs from eight different growth conditions were pooled to increase the number of detected transcripts. Furthermore, a differential RNA-Seq approach was employed for the identification of transcription start sites (TSSs). In total, 2515 TSSs were detected and classified into 834 primary (pTSS), 797 antisense (aTSS), 739 internal and 145 secondary TSSs. Our analysis of the upstream regions revealed a well conserved archaeal promoter structure. Interrogation of the distances between pTSSs and aTSSs revealed a significant number of antisense transcripts, which are a result of bidirectional transcription from the same TATA box. This mechanism of antisense transcript production could be further confirmed by in vitro transcription experiments. We assume that bidirectional transcription gives rise to non-functional antisense RNAs and that this is a widespread phenomenon in archaea due to the architecture of the TATA element and the symmetric structure of the TATA-binding protein.

Inward-facing conformation of a multidrug resistance MATE family transporter.

Multidrug and toxic compound extrusion (MATE) transporters mediate excretion of xenobiotics and toxic metabolites, thereby conferring multidrug resistance in bacterial pathogens and cancer cells. Structural information on the alternate conformational states and knowledge of the detailed mechanism of MATE transport are of great importance for drug development. However, the structures of MATE transporters are only known in V-shaped outward-facing conformations. Here, we present the crystal structure of a MATE transporter from Pyrococcus furiosus (PfMATE) in the long-sought-after inward-facing state, which was obtained after crystallization in the presence of native lipids. Transition from the outward-facing state to the inward-facing state involves rigid body movements of transmembrane helices (TMs) 2-6 and 8-12 to form an inverted V, facilitated by a loose binding of TM1 and TM7 to their respective bundles and their conformational flexibility. The inward-facing structure of PfMATE in combination with the outward-facing one supports an alternating access mechanism for the MATE family transporters.

The Transcriptional Regulator TFB-RF1 Activates Transcription of a Putative ABC Transporter in Pyrococcus furiosus.

Transcription factor B recruiting factor 1 (TFB-RF1; PF1088) is a transcription regulator which activates transcription on archaeal promoters containing weak TFB recognition elements (BRE) by recruiting TFB to the promoter. The mechanism of activation is described in detail, but nothing is known about the biological function of this protein in Pyrococcus furiosus. The protein is located in an operon structure together with the hypothetical gene pf1089 and western blot as well as end-point RT-PCR experiments revealed an extremely low expression rate of both proteins. Furthermore, conditions to induce the expression of the operon are not known. By introducing an additional copy of tfb-RF1 using a Pyrococcus shuttle vector we could circumvent the lacking expression of both proteins under standard growth conditions as indicated by western blot as well as end-point RT-PCR experiments. A ChIP-seq experiment revealed an additional binding site of TFB-RF1 in the upstream region of the pf1011/1012 operon, beside the expected target of the pf1089/tfb-RF1 region. This operon codes for a putative ABC transporter which is most-related to a multidrug export system and in vitro analysis using gel shift assays, DNase I footprinting and in vitro transcription confirmed the activator function of TFB-RF1 on the corresponding promoter. These findings are also in agreement with in vivo data, as RT-qPCR experiments also indicate transcriptional activation of both operons. Taken together, the overexpression strategy of tfb-RF1 enabled the identification of an additional operon of the TFB-RF1 regulon which indicates a transport-related function and provides a promising starting position to decipher the physiological function of the TFB-RF1 gene regulatory network in P. furiosus.

Genome-wide binding analysis of the transcriptional regulator TrmBL1 in Pyrococcus furiosus.

BACKGROUND: Several in vitro studies document the function of the transcriptional regulator TrmBL1 of Pyrococcus furiosus. These data indicate that the protein can act as repressor or activator and is mainly involved in transcriptional control of sugar uptake and in the switch between glycolysis and gluconeogenesis. The aim of this study was to complement the in vitro data with an in vivo analysis using ChIP-seq to explore the genome-wide binding profile of TrmBL1 under glycolytic and gluconeogenic growth conditions. RESULTS: The ChIP-seq analysis revealed under gluconeogenic growth conditions 28 TrmBL1 binding sites where the TGM is located upstream of coding regions and no binding sites under glycolytic conditions. The experimental confirmation of the binding sites using qPCR, EMSA, DNase I footprinting and in vitro transcription experiments validated the in vivo identified TrmBL1 binding sites. Furthermore, this study provides evidence that TrmBL1 is also involved in transcriptional regulation of additional cellular processes e.g. amino acid metabolism, transcriptional control or metabolic pathways. In the initial setup we were interested to include the binding analysis of TrmB, an additional member of the TrmB family, but western blot experiments and the ChIP-seq data indicated that the corresponding gene is deleted in our Pyrococcus strain. A detailed analysis of a new type strain demonstrated that a 16 kb fragment containing the trmb gene is almost completely deleted after the first re-cultivation. CONCLUSIONS: The identified binding sites in the P. furiosus genome classified TrmBL1 as a more global regulator as hitherto known. Furthermore, the high resolution of the mapped binding positions enabled reliable predictions, if TrmBL1 activates (binding site upstream of the promoter) or represses transcription (binding site downstream) of the corresponding genes.

Structural Insights into Nonspecific Binding of DNA by TrmBL2, an Archaeal Chromatin Protein.

The crystal structure of TrmBL2 from the archaeon Pyrococcus furiosus shows an association of two pseudosymmetric dimers. The dimers follow the prototypical design of known bacterial repressors with two helix-turn-helix (HTH) domains binding to successive major grooves of the DNA. However, in TrmBL2, the two dimers are arranged at a mutual displacement of approximately 2bp so that they associate with the DNA along the double-helical axis at an angle of approximately 80°. While the deoxyribose phosphate groups of the double-stranded DNA (dsDNA) used for co-crystallization are clearly seen in the electron density map, most of the nucleobases are averaged out. Refinement required to assume a superposition of at least three mutually displaced dsDNAs. The HTH domains interact primarily with the deoxyribose phosphate groups and polar interactions with the nucleobases are almost absent. This hitherto unseen mode of DNA binding by TrmBL2 seems to arise from nonoptimal protein-DNA contacts made by its four HTH domains resulting in a low-affinity, nonspecific binding to DNA.

The TrmB family: a versatile group of transcriptional regulators in Archaea.

Microbes are organisms which are well adapted to their habitat. Their survival depends on the regulation of gene expression levels in response to environmental signals. The most important step in regulation of gene expression takes place at the transcriptional level. This regulation is intriguing in Archaea because the eu-karyotic-like transcription apparatus is modulated by bacterial-like transcription regulators. The transcriptional regulator of mal operon (TrmB) family is well known as a very large group of regulators in Archaea with more than 250 members to date. One special feature of these regulators is that some of them can act as repressor, some as activator and others as both repressor and activator. This review gives a short updated overview of the TrmB family and their regulatory patterns in different Archaea as a lot of new data have been published on this topic since the last review from 2008.

Activation of a chimeric Rpb5/RpoH subunit using library selection.

Rpb5 is a general subunit of all eukaryotic RNA polymerases which consists of a N-terminal and a C-terminal domain. The corresponding archaeal subunit RpoH contains only the conserved C-terminal domain without any N-terminal extensions. A chimeric construct, termed rp5H, which encodes the N-terminal yeast domain and the C-terminal domain from Pyrococcus furiosus is unable to complement the lethal phenotype of a yeast rpb5 deletion strain (Δrpb5). By applying a random mutagenesis approach we found that the amino acid exchange E197K in the C-terminal domain of the chimeric Rp5H, either alone or with additional exchanges in the N-terminal domain, leads to heterospecific complementation of the growth deficiency of Δrpb5. Moreover, using a recently described genetic system for Pyrococcus we could demonstrate that the corresponding exchange E62K in the archaeal RpoH subunit alone without the eukaryotic N-terminal extension was stable, and growth experiments indicated no obvious impairment in vivo. In vitro transcription experiments with purified RNA polymerases showed an identical activity of the wild type and the mutant Pyrococcus RNA polymerase. A multiple alignment of RpoH sequences demonstrated that E62 is present in only a few archaeal species, whereas the great majority of sequences within archaea and eukarya contain a positively charged amino acid at this position. The crystal structures of the Sulfolobus and yeast RNA polymerases show that the positively charged arginine residues in subunits RpoH and Rpb5 most likely form salt bridges with negatively charged residues from subunit RpoK and Rpb1, respectively. A similar salt bridge might stabilize the interaction of Rp5H-E197K with a neighboring subunit of yeast RNA polymerase and thus lead to complementation of Δrpb5.

An extended network of genomic maintenance in the archaeon Pyrococcus abyssi highlights unexpected associations between eucaryotic homologs.

In Archaea, the proteins involved in the genetic information processing pathways, including DNA replication, transcription, and translation, share strong similarities with those of eukaryotes. Characterizations of components of the eukaryotic-type replication machinery complex provided many interesting insights into DNA replication in both domains. In contrast, DNA repair processes of hyperthermophilic archaea are less well understood and very little is known about the intertwining between DNA synthesis, repair and recombination pathways. The development of genetic system in hyperthermophilic archaea is still at a modest stage hampering the use of complementary approaches of reverse genetics and biochemistry to elucidate the function of new candidate DNA repair gene. To gain insights into genomic maintenance processes in hyperthermophilic archaea, a protein-interaction network centred on informational processes of Pyrococcus abyssi was generated by affinity purification coupled with mass spectrometry. The network consists of 132 interactions linking 87 proteins. These interactions give insights into the connections of DNA replication with recombination and repair, leading to the discovery of new archaeal components and of associations between eucaryotic homologs. Although this approach did not allow us to clearly delineate new DNA pathways, it provided numerous clues towards the function of new molecular complexes with the potential to better understand genomic maintenance processes in hyperthermophilic archaea. Among others, we found new potential partners of the replication clamp and demonstrated that the single strand DNA binding protein, Replication Protein A, enhances the transcription rate, in vitro, of RNA polymerase. This interaction map provides a valuable tool to explore new aspects of genome integrity in Archaea and also potentially in Eucaryotes.

Genetic engineering of Pyrococcus furiosus to use chitin as a carbon source.

BACKGROUND: Bioinformatic analysis of the genes coding for the chitinase in Pyrococcus furiosus and Thermococcus kodakarensis revealed that most likely a one nucleotide insertion in Pyrococcus caused a frame shift in the chitinase gene. This splits the enzyme into two separate genes, PF1233 and PF1234, in comparison to Thermococcus kodakarensis. Furthermore, our attempts to grow the wild type strain of Pyrococcus furiosus on chitin were negative. From these data we assume that Pyrococcus furiosus is most likely unable to use chitin as a carbon source. The aim of this study was to analyze in vivo if the one nucleotide insertion is responsible for the inability to grow on chitin, using a recently described genetic system for Pyrococcus furiosus. RESULTS: A marker-less genetic system for Pyrococcus furiosus was developed using simvastatin for positive selection and 6-methylpurine for negative selection. Resistance against simvastatin was achieved by overexpression of the hydroxymethylglutaryl coenzyme A reductase gene. For the resistance to 6-methylpurine the hypoxanthine-guanine phosphoribosyltransferase gene was deleted. This system was used to delete the additional nucleotide at position 1006 in PF1234. The resulting chitinase in the mutant strain was a single subunit enzyme and aligns perfectly to the enzyme from Thermococcus kodakarensis. A detailed analysis of the wild type and the mutant using counted cell numbers as well as ATP and acetate production as growth indicators revealed that only the mutant is able to use chitin as a carbon source. An additional mutant strain containing a reduced chitinase version containing just one catalytic and one chitin-binding domain showed diminished growth on chitin in comparison to the mutant containing the single large enzyme. CONCLUSIONS: Wild type Pyrococcus furiosus is most likely unable to grow on chitin in the natural biotope due to a nucleotide insertion which separates the chitinase gene into two ORFs, whereas a genetically engineered strain with the deleted nucleotide is able to grow on chitin. The overall high sequence identity of the two chitinases between P. furiosus and T. kodakarensis indicates that this mutation occurred very recently or there is still some kind of selection pressure for a functional enzyme using programmed +/-1 frameshifting.

Activation of archaeal transcription mediated by recruitment of transcription factor B.

Archaeal promoters consist of a TATA box and a purine-rich adjacent upstream sequence (transcription factor B (TFB)-responsive element (BRE)), which are bound by the transcription factors TATA box-binding protein (TBP) and TFB. Currently, only a few activators of archaeal transcription have been experimentally characterized. The best studied activator, Ptr2, mediates activation by recruitment of TBP. Here, we present a detailed biochemical analysis of an archaeal transcriptional activator, PF1088, which was identified in Pyrococcus furiosus by a bioinformatic approach. Operon predictions suggested that an upstream gene, pf1089, is polycistronically transcribed with pf1088. We demonstrate that PF1088 stimulates in vitro transcription by up to 7-fold when the pf1089 promoter is used as a template. By DNase I and hydroxyl radical footprinting experiments, we show that the binding site of PF1088 is located directly upstream of the BRE of pf1089. Mutational analysis indicated that activation requires the presence of the binding site for PF1088. Furthermore, we show that activation of transcription by PF1088 is dependent upon the presence of an imperfect BRE and is abolished when the pf1089 BRE is replaced with a BRE from a strong archaeal promoter. Gel shift experiments showed that TFB recruitment to the pf1089 operon is stimulated by PF1088, and TFB seems to stabilize PF1088 operator binding even in the absence of TBP. Taken together, these results represent the first biochemical evidence for a transcriptional activator working as a TFB recruitment factor in Archaea, for which the designation TFB-RF1 is suggested.

Shuttle vector-based transformation system for Pyrococcus furiosus.

Pyrococcus furiosus is a model organism for analyses of molecular biology and biochemistry of archaea, but so far no useful genetic tools for this species have been described. We report here a genetic transformation system for P. furiosus based on the shuttle vector system pYS2 from Pyrococcus abyssi. In the redesigned vector, the pyrE gene from Sulfolobus was replaced as a selectable marker by the 3-hydroxy-3-methylglutaryl coenzyme A reductase gene (HMG-CoA) conferring resistance of transformants to the antibiotic simvastatin. Use of this modified plasmid resulted in the overexpression of the HMG-CoA reductase in P. furiosus, allowing the selection of strains by growth in the presence of simvastatin. The modified shuttle vector replicated in P. furiosus, but the copy number was only one to two per chromosome. This system was used for overexpression of His(6)-tagged subunit D of the RNA polymerase (RNAP) in Pyrococcus cells. Functional RNAP was purified from transformed cells in two steps by Ni-NTA and gel filtration chromatography. Our data provide evidence that expression of transformed genes can be controlled from a regulated gluconeogenetic promoter.

The archaeal RNA polymerase subunit P and the eukaryotic polymerase subunit Rpb12 are interchangeable in vivo and in vitro.

The general subunit of all three eukaryotic RNA polymerases, Rpb12, and subunit P of the archaeal enzyme show sequence similarities in their N-terminal zinc ribbon and some highly conserved residues in the C-terminus. We report here that archaeal subunit P under the control of a strong yeast promoter could complement the lethal phenotype of a RPB12 deletion mutant and that subunit Rpb12 from yeast can functionally replace subunit P during reconstitution of the archaeal RNA polymerase. The DeltaP enzyme is unable to form stable open complexes, but can efficiently extend a dinucleotide on a premelted template or RNA on an elongation scaffold. This suggests that subunit P is directly or indirectly involved in promoter opening. The activity of the DeltaP enzyme can be rescued by the addition of Rpb12 or subunit P to transcription reactions. Mutation of cysteine residues in the zinc ribbon impair the activity of the enzyme in several assays and this mutated form of P is rapidly replaced by wild-type P in transcription reactions. The conserved zinc ribbon in the N-terminus seems to be important for proper interaction of the complete subunit with other RNA polymerase subunits and a 17-amino-acid C-terminal peptide is sufficient to support all basic RNA polymerase functions in vitro.

TBP domain symmetry in basal and activated archaeal transcription.

The TATA box binding protein (TBP) is the platform for assembly of archaeal and eukaryotic transcription preinitiation complexes. Ancestral gene duplication and fusion events have produced the saddle-shaped TBP molecule, with its two direct-repeat subdomains and pseudo-two-fold symmetry. Collectively, eukaryotic TBPs have diverged from their present-day archaeal counterparts, which remain highly symmetrical. The similarity of the N- and C-halves of archaeal TBPs is especially pronounced in the Methanococcales and Thermoplasmatales, including complete conservation of their N- and C-terminal stirrups; along with helix H'1, the C-terminal stirrup of TBP forms the main interface with TFB/TFIIB. Here, we show that, in stark contrast to its eukaryotic counterparts, multiple substitutions in the C-terminal stirrup of Methanocaldococcus jannaschii (Mja) TBP do not completely abrogate basal transcription. Using DNA affinity cleavage, we show that, by assembling TFB through its conserved N-terminal stirrup, Mja TBP is in effect ambidextrous with regard to basal transcription. In contrast, substitutions in either its N- or the C-terminal stirrup abrogate activated transcription in response to the Lrp-family transcriptional activator Ptr2.

The role of TrmB and TrmB-like transcriptional regulators for sugar transport and metabolism in the hyperthermophilic archaeon Pyrococcus furiosus.

TrmB of Pyrococcus furiosus was discovered as the trehalose/maltose-specific repressor for the genes encoding the trehalose/maltose high-affinity ABC transporter (the TM system). TrmB also represses the genes encoding the high affinity maltodextrin-specific ABC transporter (the MD system) with maltodextrin and sucrose as inducers. In addition, TrmB binds glucose leading to an increased repression of both, the TM and the MD system. Thus, TrmB recognizes different promoters and depending on the promoter it will be activated or inactivated for promoter binding by different sugar effectors. The TrmB-like protein TrmBL1 of P. furiosus is a global regulator and recognizes preferentially, but not exclusively, the TGM (for Thermococcales-glycolytic motif) sequence that is found upstream of the MD system as well as of genes encoding enzymes involved in the glycolytic and the gluconeogenic pathway. It responds to maltose and maltotriose as inducers and functions as repressor for the genes encoding the MD system and glycolytic enzymes, but as activator for genes encoding gluconeogenic enzymes. The TrmB-like protein TrmBL2 of P. furiosus lacks the sugar-binding domain that has been determined in TrmB. It recognizes the MD promoter, but not all TGM harboring promoters. It is evolutionary the most conserved among the Thermococcales. The regulatory range of TrmBL2 remains unclear.

Characterization of the TrmB-like protein, PF0124, a TGM-recognizing global transcriptional regulator of the hyperthermophilic archaeon Pyrococcus furiosus.

The characterization of the transcriptional regulator TrmBL1 of the hyperthermophilic archaeon Pyrococcus furiosus, homologous to TrmB (transcriptional regulator of the maltose system), was studied. The genome of P. furiosus contains three TrmB paralogues. One of the TrmB-like proteins (TrmBL), PF0124 (TrmBL1), was analysed in more detail. It regulated the expression of the genes encoding enzymes of the glycolytic pathway as well as the maltodextrin (MD) ABC transporter. By molecular sieve chromatography, purified TrmBL1 behaved at ambient temperature as a tetramer of 148.8 kDa. In the presence of 1 mM maltotriose or 5 mM maltose TrmBL1 formed octamers. As shown by electrophoretic mobility shift assay (EMSA) TrmBL1 was found to bind the MD (maltodextrin ABC transport genes) promoter DNA with sixfold higher binding affinity (K(d) 0.2 microM) than to the trehalose/maltose ABC transporter (TM) promoter (K(d) 1.2 microM). Maltotriose and maltose interfered in these assays indicating inducer function. In vitro transcription assays using purified transcription components corroborated the data obtained with EMSA and showed inhibition of transcription of the MD promoter by TrmBL1. Recently, van de Werken et al. (FEMS Microbiol Lett 2006; 260: 69-76) identified TGM, a conserved sequence (Thermococcales-Glycolytic-Motif) upstream of genes encoding glycolytic enzymes and the MD ABC transporter. The position of TGM is invariably located downstream of the BRE-TATA box and overlapping the transcription start site on each promoter. By footprint analysis TrmBL1 was found to recognize the TGM sequence in several TGM-containing promoter sequences. We identified the recognition helix in TrmBL1 revealing tyrosine (Y49) to be essential for target DNA binding. However, the TGM motif was not essential for TrmBL1 binding. We conclude that TrmBL1 is a global sugar-sensing transcriptional regulator controlling the genes of transport systems and of sugar-metabolizing enzymes.