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from Pyrococcus furiosus (9, 10), Lrs-14 from Sulfolobus solfataricus (11), and Ptr1 from Methanococcus jannaschii (unpublished results), have only been shown to repress transcription by their cognate RNA polymerases.Here we show that Ptr2, a site-specific helix-turn-helix DNAbinding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...

SummaryThe relatively complex archaeal RNA polymerases are constructed along eukaryotic lines, and require two initiation factors for promoter recognition and specific transcription that are homologues of the RNA polymerase II TATA-binding protein and TFIIB. Many archaea also produce histones. In contrast, the transcriptional regulators encoded by archaeal genomes are primarily of bacterial rather than eukaryotic type. It is this combination of elements commonly regarded as separate and mutually exclusive that promises unifying insights into basic transcription mechanisms across all three domains of life.

Development of bacteriophage T4 in Escherichia coli requires the sequential recognition of three classes of promoters: early, middle, and late. Recognition of middle promoters is known to require the motA gene product, a protein that binds specifically to the "Mot box" located at the -30 region of these promoters. In vivo, the asiA gene product is as critical for middle mode RNA synthesis as is that of the motA gene. In vitro, AsiA protein is known to loosen the r70-core RNA polymerase interactions and to inhibit some oJ70-dependent transcription, presumably through binding to the cr7O subunit. Here we show that, in vitro, purified MotA and AsiA proteins are both necessary and sufficient to activate transcription initiation at T4 middle promoters by the E. coli RNA polymerase in a o-70-dependent manner. AsiA is also shown to inhibit recognition of T4 early promoters and may play a pivotal role in the recognition of all three classes of phage promoters.In the course of phage T4 development in Escherichia coli, all phage gene transcripts are synthesized by the host RNA polymerase (RNAP), whose structure and functional properties are modified by phage-coded proteins, leading to the sequential recognition of three different classes of promoters: early, middle, and late. The early promoters are recognized immediately after infection by unmodified host RNAP even when phage protein synthesis is inhibited. They are also recognized in vitro on T4 DNA by purified E. coli RNAP holoenzyme (a2313' a-70). During this early period, the host RNAP undergoes several T4-induced changes (1). Its a subunits are ADP-ribosylated (by the T4 alt and mod gene products), and it becomes tightly associated with at least two small T4 proteins, RpbA and AsiA. By 3 min after infection (at 30WC), some, if not all, of these early promoters are turned off (2, 3), but the mechanism of this transcriptional shutoff has not yet been elucidated. At about the same time, transcription from middle promoters is turned on. These promoters have a conserved "Mot box" sequence, 5'-(a/t)(a/t)TGCTT(t/c)A-3', centered around bp -30, 11-13 bases upstream of a standard E. coli -10 consensus sequence (4-6). The T4 late promoters have simpler sequence determinants, consisting primarily of the octamer TATAAATA at the -10 region. Transcription from these late promoters requires, among other things, the phage cr factor gp55 (7). In vitro, the E. coli a-70 has been shown to be dominant over gp55 in competition for core RNAP and reduces late promoter recognition (8,9). Yet, during the late period of T4 development, a-70 and gp55 coexist (and apparently cofunction) in the infected cell (10, 11).The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 1451Recognition of T4 middle promoters requires the early gene product MotA. MotA protein has been purified and is known to bind specifically to the -30 Mot box seque...

The anti-&0 factor of bacteriophage T4 is a 10-kDa (10K) protein which inhibits the o70-directed initiation of transcription by Escherichia coli RNA polymerase holoenzyme. We have partially purified the anti-&0 factor and obtained the sequence of a C-terminal peptide of this protein. Using reverse genetics, we have identified, at the end of the lysis gene t and downstream of an as yet unassigned phage T4 early promoter, an open reading frame encoding a 90-amino-acid protein with a predicted molecular weight of 10,590. This protein has been overproduced in a phage T7 expression system and partially purified. It shows a strong inhibitory activity towards &70-directed transcription (by RNA polymerase holoenzyme), whereas it has no significant effect on o70-independent transcription (by RNA polymerase core enzyme). At high ionic strength, this inhibition is fully antagonized by the neutral detergent Triton X-100. Our results corroborate the initial observations on the properties of the phage T4 10K anti-o70 factor, and we therefore propose that the gene which we call asi4, identified in the present study, corresponds to the gene encoding this T4 transcriptional inhibitor.During infection of Escherichia coli by phage T4, a large part of the program of viral gene expression is regulated at the transcriptional level. The host's RNA polymerase transcribes the 200 or so viral genes from different classes of T4-specific promoters (23). Some of these classes are recognized only after T4-coded functions modify this enzyme's specificity. The a subunits of RNA polymerase are covalently modified by ADP-ribosylation, and the enzyme binds a series of phage-coded proteins (13). Among these, the products of genes 33, 45, and 55 are required for late transcription (12). In particular, gp 55 (16) redirects RNA polymerase transcription initiation from T4 late promoters by replacing the E. coli c70 subunit. The product of gene rpbA (45) strongly binds to RNA polymerase core. A smaller RNA polymerase-binding gene product, the 10-kDa (10K) protein, copurifies with au7 on phosphocellulose and inhibits o70-directed transcription initiation by E. coli RNA polymerase holoenzyme (36). It is believed that the interaction of the T4 10K protein with bacterial c70 weakens o&0-core interaction; this, in turn, would allow gp 55 to successfully compete for core enzyme during T4 development (12). The 10K protein found in lysates from T4-infected cells strongly binds to RNA polymerase agarose affinity columns (27). Starting from this observation, we have previously used a biochemical approach to identify the gene encoding the T4 10K protein (25) T4 (36). This approach to the partial purification of gp asiA was chosen to minimize its progressive loss, observed during the purification of RNA polymerase from T4-infected cells (35). In Fig. 1, we show the autoradiography of an SDS-polyacrylamide gel illustrating the separation of the RNA polymerase-binding proteins coded by phage T4 after Bio-Rex 70 chromatography. The gp asiA is essentially found in...