The hyperthermophilic archaeon Methanococcus jannaschii encodes two putative transcription regulators, Ptr1 and Ptr2, that are members of the Lrp/AsnC family of bacterial transcription regulators. In contrast, this archaeon's RNA polymerase and core transcription factors are of eukaryotic type. Using the M. jannaschii high-temperature in vitro transcription system, we show that Ptr2 is a potent transcriptional activator, and that it conveys its stimulatory effects on its cognate eukaryal-type transcription machinery from an upstream activating region composed of two Ptr2-binding sites. Transcriptional activation is generated, at least in part, by Ptr2-mediated recruitment of the TATA-binding protein to the promoter.T he core components of archaeal transcription closely resemble those of eukaryotic RNA polymerase II (1). Archaeal promoters consist of an AϩT-rich TATA-like element recognized by archaeal TATA-binding protein (TBP); the TFIIBrelated transcription factor B (TFB) binds to the TBP-DNA complex and directs a eukaryotic-type RNA polymerase (RNAP) to specifically initiate transcription at an initiator sequence located some 25 bp downstream of the TATA element. Efficient preinitiation complex (PIC) assembly is ensured by the adjacent purine-rich BRE element, which mediates sequencespecific interactions with TFB upstream of the TATA box (2) and dictates the directionality of transcription complex assembly and initiation (3). TBP, TFB, and RNAP are necessary and sufficient to direct transcription at many archaeal promoters in vitro; a modest stimulatory effect of TFE, the archaeal homologue of the ␣ subunit of the RNA polymerase II transcription factor TFIIE, is discerned under conditions of suboptimal TBP-TATA box interaction (4, 5).On the other hand, all archaeal genomes sequenced to date encode potential transcription regulators of bacterial type, underscoring the chimeric nature of the archaeal transcription apparatus (6, 7). Particular interest is attached to the question of how these bacterial-type effectors, especially activators, generate regulation of a eukaryote-like transcription system. All of the putative regulators of transcription that have been characterized in vitro, the metal-dependent repressor 1 (MDR1) from Archaeoglobus fulgidus (8), as well as the homologues LrpA from Pyrococcus furiosus (9, 10), Lrs-14 from Sulfolobus solfataricus (11), and Ptr1 from Methanococcus jannaschii (unpublished results), have only been shown to repress transcription by their cognate RNA polymerases.Here we show that Ptr2, a site-specific helix-turn-helix DNAbinding protein from the hyperthermophilic archaeon M. jannaschii and homologue of the bacterial leucine-responsive regulatory protein (Lrp) family of transcription factors, is a potent transcriptional activator in vitro. We also show that Ptr2 conveys its stimulatory effects on its cognate transcription machinery through direct recruitment of TBP. Materials and MethodsProtein Purification. The RNA polymerase from Methanococcus͞ Methanocaldococcus jannaschi...

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.Several reports addressed the initial establishment of genetic techniques for the Thermococcales, a major group of hyperthermophilic archaea including the genera Thermococcus and Pyrococcus. The first experiments described used the plasmid pGT5 from Pyrococcus abyssi. This plasmid is only 3,440 bp in size and replicates via a rolling circle mechanism (7). The archaeal plasmid was fused with a pUC19 vector to create a potential shuttle vector between Escherichia coli and Pyrococcus furiosus (1). This construct could be transformed in both organisms by CaCl 2 treatment. Later, this construct was modified by introducing the alcohol dehydrogenase gene from Sulfolobus solfataricus as a selectable marker (3). The resulting plasmids pAG1 and pAG2 were maintained for several generations in E. coli, in the euryarchaeote P. furiosus, and also in the crenarchaeote Sulfolobus acidocaldarius. The presence of these plasmids in the two archaea conferred resistance to butanol and benzyl alcohol.As the attempts to use this selection system for P. abyssi failed, a new shuttle vector, pYS2, was created (17). This construct is also based on the archaeal pGT5 plasmid and a bacterial vector, pLitmus38. It contains the pyrE gene of S. acidocaldarius, a key enzyme of the pyrimidine biosynthetic pathway, as a selectable marker. For the transformation procedure, a Pyrococcus strain was used containing a pyrE mutation which led to a uracil-auxotrophic phenotype. Using the shuttle vector pYS2 in combination with a polyethylene glycolspheroplast method, it was possible to transform the pyrE mutant of P. abyssi to uracil prototrophy. Although the transformation frequency was very low, the shuttle vector was stably maintained at high copy number under selective conditions in both E. coli and P. abyssi (17).A major breakthrough in the establishment of genetic tools for hyperthermophilic euryar...

We reported previously that cell-free transcription in the Archaea Methanococcus and Pyrococcus depends upon two archaeal transcription factors, archaeal transcription factor A (aTFA) and archaeal transcription factor B (aTFB). In the genome of Pyrococcus genes encoding putative homologues of eucaryal transcription factors TATA-binding protein (TBP) and TFIIB have been detected. Here, we report that Escherichia coli synthesized Pyrococcus homologues of TBP and TFIIB are able to replace endogenous aTFB and aTFA in cellfree transcription reactions. Antibodies raised against archaeal TBP and TFIIB bind to polypeptides of identical molecular mass in the aTFB and aTFA fraction. These data identify aTFB as archaeal TBP and aTFA as the archaeal homologue of TFIIB. At the Pyrococcus glutamate dehydrogenase (gdh) promoter these two bacterially produced transcription factors and endogenous RNA polymerase are sufficient to direct accurate and active initiation of transcription. DNase I protection experiments revealed Pyrococcus-TBP producing a characteristic footprint between position ؊20 and ؊34 centered around the TATA box of gdh promoter. Pyrococcus-TFIIB did not bind to the TATA box but bound cooperatively with Pyrococcus-TBP generating an extended DNase I footprinting pattern ranging from position ؊19 to ؊42. These data suggest that the Pyrococcus homologue of TFIIB associates with the TBP-promoter binary complex as its eucaryal counterpart, but in contrast to eucaryal TFIIB, it causes an extension of the protection to the region upstream of the TATA box.Recent work established that cell-free transcription in Archaea is mediated by transcription factors (1-3). In the Euryarchaeon (4) Methanococcus two distinct archaeal transcription factors aTFA 1 and aTFB have been identified (1, 5). Highly purified Methanococcus aTFB showed striking similarities to eucaryal TATA-binding proteins (TBP). It exists as a dimer in solution (5), can be replaced by yeast and human TBPs in cell-free transcription reactions (6), and binds in mobility gel shift assays (7) to DNA fragments harboring an archaeal (8, 9) or eucaryal TATA box. Furthermore, the protein translation of a putative TBP homologue encoded in the genome of Thermococcus (10) was able to substitute for Methanococcus aTFB in cell-free transcription reactions and showed serological crossreaction with this polypeptide (11). Owing to its low stability purification of the aTFA activity thus far was not possible, but the incubation of aTFB or eucaryal TBPs in combination with aTFA results in template commitment (7, 6), suggesting that it binds to and stabilizes the aTFB-promoter complex.We have recently described a cell-free transcription system for the hyperthermophilic Archaeon Pyrococcus furiosus (12). In this system, specific transcription was as well dependent upon the presence of aTFB and aTFA activities. The discovery of two genes encoding putative homologues of eucaryal TBP and RNA polymerase II transcription factor B (TFIIB) in the genome of Pyrococcus (13-15) prompted u...

SummaryTrmB is the transcriptional repressor for the gene cluster of the trehalose/maltose ABC transporter of the hyperthermophilic archaea Thermococcus litoralis and Pyrococcus furiosus ( malE or TM operon), with maltose and trehalose acting as inducers. We found that TrmB (the protein is identical in both organisms) also regulated the transcription of genes encoding a separate maltodextrin ABC transporter in P. furiosus ( mdxE or MD operon) with maltotriose, longer maltodextrins and sucrose acting as inducers, but not with maltose or trehalose. In vitro transcription of the malE and the mdxE operons was inhibited by TrmB binding to the different operator sequences. Inhibition of the TM operon was released by maltose and trehalose whereas inhibition of the MD operon was released by maltotriose and larger maltodextrins as well as by sucrose. Scanning mutagenesis of the TM operator revealed the role of the palindromic TACTNNNAGTA sequence for TrmB recognition. TrmB exhibits a broad spectrum of sugar-binding specificity, binding maltose, sucrose, maltotriose and trehalose in decreasing order of affinity, half-maximal binding occurring at 20, 60, 250 and 500 m m m m M substrate concentration respectively. Of all substrates, only maltose shows sigmoidal binding characteristics with a Hill coefficient of 2. As measured by molecular sieve chromatography and cross-linking TrmB behaved as dimer in dilute buffer solution at room temperature. We conclude that TrmB acts as a bifunctional transcriptional regulator acting on two different promoters and being differentially controlled by binding to different sugars. We believe this to represent a novel strategy of prokaryotic transcription regulation.

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.

We describe here the establishment of a cell-free transcription system for the hyperthermophilic Archaeon Pyrococcus furiosus using the cloned glutamate dehydrogenase (gdh) gene as template. The in vitro system that operated up to a temperature of 85 degrees C initiated transcription 23 bp downstream of a TATA box located 45 bp upstream of the translational start codon of gdh mRNA, at the same site as in Pyrococcus cells. Mutational analyses revealed that this TATA box is essential for in vitro initiation of transcription. Pyrococcus transcriptional components were separated into at least two distinct transcription factor activities and RNA polymerase. One of these transcription factors could be functionally replaced by Methanococcus aTFB and Thermococcus TATA bind- ing protein (TBP). Immunochemical analyses demonstrated a structural relationship between Pyrococcus aTFB and Thermococcus TBP. These findings indicate that a TATA box and a TBP are essential components of the Pyrococcus transcriptional machinery.

We have analyzed the function of an archaeal protein (now called transcription factor S (TFS)) that shows sequence similarity to eukaryotic transcription factor IIS (TFIIS) as well as to small subunits of eukaryotic RNA polymerases I (A12.6), II (B12.2), and III (C11). Western blot analysis with antibodies against recombinant TFS demonstrated that this protein is not a subunit of the RNA polymerase. In vitro transcription experiments with paused elongation complexes at position ؉25 showed that TFS is able to induce cleavage activity in the archaeal RNA polymerase in a similar manner to TFIIS. In the presence of TFS, the cleavage activity of the RNA polymerase truncates the RNA back to position ؉15 by releasing mainly dinucleotides from the 3 -end of the nascent RNA. Furthermore, TFS reduces the amount of non-chaseable elongation complexes at position ؉25 as well as position ؉45. These findings clearly demonstrate that this protein has a similar function to eukaryotic TFIIS.Considerable progress has been made recently in the functional analysis of initiation of transcription in Archaea. It seems to be clear now that initiation of transcription in Archaea is mediated by two basal transcription factors, archaeal TATA box-binding protein (aTBP) 1 and TFB, which are orthologous to the eukaryotic TATA box-binding protein and TFIIB (reviewed in Refs. 1-3). These two factors together with RNA polymerase are necessary and sufficient for initiation of in vitro transcription at some promoters (4, 5). These findings are in line with the data of genome sequencing projects in Archaea. Analysis of the genomes of Methanococcus jannaschii, Methanobacterium thermoautotrophicum, Pyrococcus horikoshii, and Archaeoglobus fulgidus indicated that there are, with the exception of a putative ␣-subunit of TFIIE, no further eukaryote-like initiation factors corresponding to TFIIA, TFIIF, or TFIIH (6 -9). Interestingly the genome sequences revealed the presence of a putative transcription factor, which could be involved in the elongation of transcription.The sequence of this protein was first identified in Sulfolobus acidocaldarius and was suggested to be a homologue to the eukaryotic elongation factor TFIIS (10). Identification of an additional candidate in Thermococcus celer and a detailed analysis of the sequence similarity of these proteins to eukaryotic counterparts revealed that the archaeal TFIIS-like protein is much more likely to be a subunit of the archaeal RNA polymerase than a transcription factor with a similar function to eukaryotic TFIIS (3,(11)(12)(13)(14). Several lines of evidence support this suggestion. First, the archaeal genes code for proteins that are ϳ120 amino acids in size, whereas the eukaryotic TFIIS proteins are much larger (ϳ300 amino acids). Second, the archaeal proteins contain two putative zinc-binding domains instead of the single domain found in eukaryotic TFIIS proteins. Third, eukaryotic RNA pol I, II, and III contain related subunits (A12.6, B12.2, and C11) of similar size and two zincbinding motifs. ...

SummaryThe archaeal transcription apparatus is chimeric: its core components (RNA polymerase and basal factors) closely resemble those of eukaryotic RNA polymerase II, but the putative archaeal transcriptional regulators are overwhelmingly of bacterial type. Particular interest attaches to how these bacterial-type effectors, especially activators, regulate a eukaryotelike transcription system. The hyperthermophilic archaeon Methanocaldococcus jannaschii encodes a potent transcriptional activator, Ptr2, related to the Lrp/AsnC family of bacterial regulators. Ptr2 activates rubredoxin 2 ( rb2 ) transcription through a bipartite upstream activating site (UAS), and conveys its stimulatory effects on its cognate transcription machinery through direct recruitment of the TATA binding protein (TBP). A functional dissection of the highly constrained architecture of the rb2 promoter shows that a 'one-site' minimal UAS suffices for activation by Ptr2, and specifies the required placement of this site. The presence of such a simplified UAS upstream of the natural rubrerythrin ( rbr ) promoter also suffices for positive regulation by Ptr2 in vitro , and TBP recruitment remains the primary means of transcriptional activation at this promoter.