Mohamed Ouhammouch

Archaeal transcription and its regulators

The 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.

Activation of archaeal transcription by recruitment of the TATA-binding protein

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 archaeons 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.

A thermostable platform for transcriptional regulation: the DNA-binding properties of two Lrp homologs from the hyperthermophilic archaeon Methanococcus jannaschii

The hyperthermophilic archaeon Methanococcus jannaschii encodes two putative transcription regulators, Ptr1 and Ptr2, related to the bacterial Lrp/AsnC family of transcriptional regulators. We show that these two small helix–turn–helix proteins are specific DNA‐binding proteins recognizing sites in their respective promoter regions. In vitro selection at high temperature has been used to isolate sets of high‐ affinity DNA sites that define a palindromic consensus binding sequence for each protein. Ptr1 and Ptr2 bind these cognate sites from one side of the DNA helix, as dimers, with each protein monomer making base‐ specific contacts in the major groove. As the first archaeal DNA‐binding proteins with clearly defined specificities, Ptr1 and Ptr2 provide a thermostable DNA‐binding platform for analysis of effector interactions with the core archaeal transcription apparatus; a platform allowing manipulation of promoter structure and examination of mechanisms of action at heterologous promoters.

Promoter architecture and response to a positive regulator of archaeal transcription

The 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 eukaryote‐like 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.

The mechanism of transcriptional activation by the topologically DNA-linked sliding clamp of bacteriophage T4.

Three viral proteins participate directly in transcription of bacteriophage T4 late genes: the sigma-family protein gp55 provides promoter recognition, gp33 is the co-activator, and gp45 is the activator of transcription; gp33 also represses transcription in the absence of gp45. Transcriptional activation by gp45, the toroidal sliding clamp of the T4 DNA polymerase holoenzyme, requires assembly at primer-template junctions by its clamp loader. The mechanism of transcriptional activation has been analyzed by examining rates of formation of open promoter complexes. The basal gp55-RNA polymerase holoenzyme is only weakly held in its initially formed closed promoter complex, which subsequently opens very slowly. Activation ( approximately 320-fold in this work) increases affinity in the closed complex and accelerates promoter opening. Promoter opening by gp55 is also thermo-irreversible: the T4 late promoter does not open at 0 degrees C, but once opened at 30 degrees C remains open upon shift to the lower temperature. At a hybrid promoter for sigma(70) and gp55-holoenzymes, only gp55 confers thermo-irreversibility of promoter opening. Interaction of gp45 with a C-terminal epitope of gp33 is essential for the co-activator function of gp33.

An expanding family of archaeal transcriptional activators

Transcriptional regulation in the archaea involves a mosaic of DNA-binding proteins frequently (although not exclusively) of bacterial type, modulating a eukaryal-type core transcription apparatus. Methanocaldococcus jannaschii (Mja) Ptr2, a homologue of the Lrp/AsnC family of bacterial transcription regulators that are among the most widely disseminated archaeal DNA-binding proteins, has been shown to activate transcription by its conjugate hyperthermophilic RNA polymerase. Here, two in vitro systems have been exploited to show that Ptr2 and a Lrp homologue from the thermophile Methanothermococcus thermolithotrophicus (Mth) activate transcription over a ≈40°C range, in conjunction with their cognate TATA-binding proteins (TBPs) and with heterologous TBPs. A closely related homologue from the mesophile Methanococcus maripaludis (Mma) is nearly inert as a transcriptional activator, but a cluster of mutations that converts a surface patch of Mma Lrp to identity with Ptr2 confers transcriptional activity. Mja, Mth, and Mma TBPs are interchangeable for basal transcription, but their ability to support Lrp-mediated transcriptional activation varies widely, with Mja TBP the most active and Mth TBP the least active partner. The implications of this finding for understanding the roles of TBP paralogues in supporting the gene-regulatory repertoires of archaeal genomes are briefly noted.

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.

Hybrid Ptr2‐like activators of archaeal transcription

Methanocaldococcus jannaschii Ptr2, a member of the Lrp/AsnC family of bacterial DNA‐binding proteins, is an activator of its eukaryal‐type core transcription apparatus. In Lrp‐family proteins, an N‐terminal helix–turn–helix DNA‐binding and dimerizing domain is joined to a C‐terminal effector and multimerizing domain. A cysteine‐scanning surface mutagenesis shows that the C‐terminal domain of Ptr2 is responsible for transcriptional activation; two types of DNA binding‐positive but activation‐defective mutants are found: those unable to recruit the TBP and TFB initiation factors to the promoter, and those failing at a post‐recruitment step. Transcriptional activation through the C‐terminal Ptr2 effector domain is exploited in a screen of other Lrp effector domains for activation capability by constructing hybrid proteins with the N‐terminal DNA‐binding domain of Ptr2. Two hybrid proteins are effective activators: Ptr–H10, fusing the effector domain of Pyrococcus furiosus LrpA, and Ptr–H16, fusing the P. furiosus ORF1231 effector domain. Both new activators exhibit distinguishing characteristics: unlike octameric Ptr2, Ptr–H10 is a dimer; unlike Ptr2, the octameric Ptr–H16 poorly recruits TBP to the promoter, but more effectively co‐recruits TFB with TBP. In contrast, the effector domain of Ptr1, the M. jannaschii Ptr2 paralogue, yields only very weak activation.