Crystal structure of the bacteriophage T4 late-transcription coactivator gp33 with the ß-subunit flap domain of Escherichia coli RNA polymerase.

Activated transcription of the bacteriophage T4 late genes, which is coupled to concurrent DNA replication, is accomplished by an initiation complex containing the host RNA polymerase associated with two phage-encoded proteins, gp55 (the basal promoter specificity factor) and gp33 (the coactivator), as well as the DNA-mounted sliding-clamp processivity factor of the phage T4 replisome (gp45, the activator). We have determined the 3.0 Å-resolution X-ray crystal structure of gp33 complexed with its RNA polymerase binding determinant, the ß-flap domain. Like domain 4 of the promoter specificity σ factor (σ(4)), gp33 interacts with RNA polymerase primarily by clamping onto the helix at the tip of the ß-flap domain. Nevertheless, gp33 and σ(4) are not structurally related. The gp33/ß-flap structure, combined with biochemical, biophysical, and structural information, allows us to generate a structural model of the T4 late promoter initiation complex. The model predicts protein/protein interactions within the complex that explain the presence of conserved patches of surface-exposed residues on gp33, and provides a structural framework for interpreting and designing future experiments to functionally characterize the complex.

Transcription of the T4 late genes.

This article reviews the current state of understanding of the regulated transcription of the bacteriophage T4 late genes, with a focus on the underlying biochemical mechanisms, which turn out to be unique to the T4-related family of phages or significantly different from other bacterial systems. The activator of T4 late transcription is the gene 45 protein (gp45), the sliding clamp of the T4 replisome. Gp45 becomes topologically linked to DNA through the action of its clamp-loader, but it is not site-specifically DNA-bound, as other transcriptional activators are. Gp45 facilitates RNA polymerase recruitment to late promoters by interacting with two phage-encoded polymerase subunits: gp33, the co-activator of T4 late transcription; and gp55, the T4 late promoter recognition protein. The emphasis of this account is on the sites and mechanisms of actions of these three proteins, and on their roles in the formation of transcription-ready open T4 late promoter complexes.

E. Peter Geiduschek.

Peter Geiduschek was an undergraduate Chemistry major at Columbia University and received his Physical Chemistry Ph.D. at Harvard in 1952 for research under the direction of Paul Doty. After short stints teaching chemistry at Yale and the University of Michigan, and an early two-year sabbatical asa US Army draftee, he came to the University of Chicago's Committee on Biophysics, where he was first introduced to enzymology and to phage. In 1970, he joined the Department of Biology of the then relatively new University of California campus at La Jolla, and has remained at UCSD since. His research contributions have primarily dealt with mechanisms of transcription and gene regulation, pursued in the specific microbial context of phage-infected bacteria, eukaryotes (budding yeast and RNA polymerase III) and archaea.

Transcriptional activation in the context of repression mediated by archaeal histones.

Many archaea (including all the methanogens, nearly all euryarchaeotes, and some crenarchaeotes) use histones as components of the chromatin that compacts their genomes. The archaeal histones are homo- and heterodimers that pair on DNA to form tetrasomes (as the eukaryotic histones H3 and H4 do). The resulting DNA packaging is known to interfere with assembly of the archaeal transcription apparatus at promoters; the ability of transcriptional activation to function in repressive archaeal chromatin has not yet been explored in vitro. Using four of the Methanocaldococcus jannaschii (Mja) histones, we have examined activation of the model Mja rb2 transcription unit by the Mja transcriptional activator Ptr2 in this simplified-chromatin context. Using hydroxyl radical footprinting, we find that the Ptr2-specific rb2 upstream activating site is a preferred histone-localizing site that nucleates histone: DNA-binding radiating from the rb2 promoter. Nevertheless, Ptr2 competes effectively with histones for access to the rb2 promoter and most potently activates transcription in vitro at histone concentrations that extensively coat DNA and essentially silence basal transcription.

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.

Without a license, or accidents waiting to happen.

This is a memoir of circumstances that have shaped my life as a scientist, some of the questions that have excited my interest, and some of the people with whom I have shared that pursuit. I was introduced to transcription soon after the discovery of RNA polymerase and have been fascinated by questions relating to gene regulation since that time. My account touches on early experiments dealing with the ability of RNA polymerase to selectively transcribe its DNA template. Temporal programs of transcription that control the multiplication cycles of viruses (phages) and the precise mechanisms generating this regulation have been a continuing source of fascination and new challenges. A longtime interest in eukaryotic RNA polymerase III has centered on yeast and on the enumeration and properties of its transcription initiation factors, the architecture of its promoter complexes, and the mechanism of transcriptional initiation. These areas of research are widely regarded as separate, but to my thinking they have posed similar questions, and I have been unwilling or unable to abandon either one for the other. An additional interest in archaeal transcription can be seen as stemming naturally from this point of view.

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.

Dissection of the bacteriophage T4 late promoter complex.

Activated transcription of the bacteriophage T4 late genes is generated by a mechanism that stands apart from the common modalities of transcriptional regulation: the activator is gp45, the viral replisome's sliding clamp; two sliding-clamp-binding proteins, gp33 and gp55, replace the host RNA polymerase (RNAP) sigma subunit. We have mutagenized, reconfigured and selectively disrupted individual interactions of the sliding clamp with gp33 and gp55 and have monitored effects on transcription. The C-terminal sliding-clamp-binding epitopes of gp33 and gp55 are perfectly interchangeable, but the functions of these two RNAP-sliding clamp connections differ: only the gp33-gp45 linkage is essential for activation, while loss of the gp55-gp45 linkage impairs but does not abolish activation. Formation of transcription-ready promoter complexes by the sliding-clamp-activated wild-type T4 RNAP resists competition by high concentrations of the polyanion heparin. This avid formation of promoter complexes requires both linkages of the T4 late RNAP to the sliding clamp. Preopening the promoter compensates for loss of the gp55-gp45 but not the gp33-gp45 linkage. We interpret the relationship of these findings and our prior analysis to the common model of transcriptional initiation in bacteria in terms of two parallel pathways, with two RNAP holoenzymes and two DNA templates: (1) gp55-RNAP and the T4 late promoter execute basal transcription; (2) gp55-gp33-RNAP and the T4 late promoter with its mobile enhancer, the T4 sliding clamp, execute activated transcription. gp55 and gp33 perform sigma-like functions, gp55 in promoter recognition and gp33 (as well as gp55) in enhancer recognition. gp33 operates the switch between these two pathways by repressing basal transcription.

Transcription: adjusting to adversity by regulating RNA polymerase.

Under growth-limiting conditions, budding yeast shut down transcription of genes of the translation apparatus. Recent studies have shown that this response is signaled, in part, by multiple pathways that converge on Maf1, leading to a change of this protein's phosphorylation state and its relocation to the nucleus, where it represses RNA polymerase III.

The role of an upstream promoter interaction in initiation of bacterial transcription.

The bacterial RNA polymerase (RNAP) recognizes promoters through sequence-specific contacts of its promoter-specificity components (sigma) with two DNA sequence motifs. Contacts with the upstream ('-35') promoter motif are made by sigma domain 4 attached to the flap domain of the RNAP beta subunit. Bacteriophage T4 late promoters consist solely of an extended downstream ('-10') motif specifically recognized by the T4 gene 55 protein (gp55). Low level basal transcription is sustained by gp55-RNAP holoenzyme. The late transcription coactivator gp33 binds to the beta flap and represses this basal transcription. Gp33 can also repress transcription by Escherichia coli sigma70-RNAP holoenzyme mutated to allow gp33 access to the beta flap. We propose that repression is due to gp33 blocking an upstream sequence-independent DNA-binding site on RNAP (as sigma70 domain 4 does) but, unlike sigma70 domain 4, providing no new DNA interaction. We show that this upstream interaction is essential only at an early step of transcription initiation, and discuss the role of this interaction in promoter recognition and transcriptional regulation.

Mapping the principal interaction site of the Brf1 and Bdp1 subunits of Saccharomyces cerevisiae TFIIIB.

The Brf1 subunit of the central RNA polymerase (pol) III transcription initiation factor TFIIIB is bipartite; its N-terminal TFIIB-related half is principally responsible for recruiting pol III to the promoter and for promoter opening near the transcriptional start site, whereas its pol III-specific C-terminal half contributes most of the affinities that hold the three subunits of TFIIIB together. Here, the principal attachment site of Brf1 for the Bdp1 subunit of TFIIIB has been mapped by a combination of structure-informed, site-directed mutagenesis and photochemical protein-DNA cross-linking. A 66-amino acid segment of Brf1 is shown to serve as a two-sided adhesive surface, with the side chains projecting away from its extended interface with TATA-binding protein anchoring Bdp1 binding. An extensive collection of N-terminal, C-terminal, and internal deletion proteins has been used to demarcate the interacting Bdp1 domain to a 66-amino acid segment that includes the SANT domain of this subunit and is phylogenetically the most conserved region of Bdp1.

Reconfiguring the connectivity of a multiprotein complex: fusions of yeast TATA-binding protein with Brf1, and the function of transcription factor IIIB.

Transcription factor (TF) IIIB, the central transcription initiation factor of RNA polymerase III (pol III), is composed of three subunits, Bdp1, Brf1 and TATA-binding protein (TBP), all essential for normal function in vivo and in vitro. Brf1 is a modular protein: Its N-proximal half is related to TFIIB and binds similarly to the C-terminal stirrup of TBP; its C-proximal one-third provides most of the affinity for TBP by binding along the entire length of the convex surface and N-terminal lateral face of TBP. A structure-informed triple fusion protein, with TBP core placed between the N- and C-proximal domains of Brf1, has been constructed. The Brf1-TBP triple fusion protein effectively replaces both Brf1 and TBP in TFIIIC-dependent and -independent transcription in vitro, and forms extremely stable TFIIIB-DNA complexes that are indistinguishable from wild-type TFIIIB-DNA complexes by chemical nuclease footprinting. Unlike Brf1 and TBP, the triple fusion protein is able to recruit pol III for TATA box-directed transcription of linear and supercoiled DNA in the absence of Bdp1. The Brf1-TBP triple fusion protein also effectively replaces Brf1 function in vivo as the intact protein, creating a TBP paralogue in yeast that is privatized for pol III transcription.

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 approximately 40 degrees 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.

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.

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 bacteriophage T4 late-transcription coactivator gp33 binds the flap domain of Escherichia coli RNA polymerase.

Transcription of bacteriophage T4 late genes requires concomitant DNA replication. T4 late promoters, which consist of a single 8-bp -10 motif, are recognized by a holoenzyme containing Escherichia coli RNA polymerase core and the T4-encoded promoter specificity subunit, gp55. Initiation of transcription at these promoters by gp55-holoenzyme is inefficient, but is greatly activated by the DNA-loaded DNA polymerase sliding clamp, gp45, and the coactivator, gp33. We report that gp33 attaches to the flap domain of the Escherichia coli RNA polymerase beta-subunit and that this interaction is essential for activation. The beta-flap also mediates recognition of -35 promoter motifs by binding to sigma(70) domain 4. The results suggest that gp33 is an analogue of sigma(70) domain 4 and that gp55 and gp33 together constitute two parts of the T4 late sigma. We propose a model for the role of the gp45 sliding clamp in activation of T4 late-gene transcription.

A fully recombinant system for activator-dependent archaeal transcription.

The core components of the archaeal transcription apparatus closely resemble those of eukaryotic RNA polymerase II, while the DNA-binding transcriptional regulators are predominantly of bacterial type. Here we report the construction of an entirely recombinant system for positively regulated archaeal transcription. By omitting individual subunits, or sets of subunits, from the in vitro assembly of the 12-subunit RNA polymerase from the hyperthermophile Methanocaldococcus jannaschii, we describe a functional dissection of this RNA polymerase II-like enzyme, and its interactions with the general transcription factor TFE, as well as with the transcriptional activator Ptr2.

Topography of the euryarchaeal transcription initiation complex.

Transcription in the Archaea is carried out by RNA polymerases and transcription factors that are highly homologous to their eukaryotic counterparts, but little is known about the structural organization of the archaeal transcription complex. To address this, transcription initiation complexes have been formed with Pyrococcus furiosus transcription factors (TBP and TFB1), RNA polymerase, and a linear DNA fragment containing a strong promoter. The arrangement of proteins from base pair -35 to +20 (relative to the transcriptional start site) has been analyzed by photochemical protein-DNA cross-linking. TBP cross-links to the TATA box and TFB1 cross-links both upstream and downstream of the TATA box, as expected, but the sites of most prominent TFB1 cross-linking are located well downstream of the TATA box, reaching as far as the start site of transcription, suggesting a role for TFB1 in initiation of transcription that extends beyond polymerase recruitment. These cross-links indicate the transcription factor orientation in the initiation complex. The pattern of cross-linking of four RNA polymerase subunits (B, A', A", and H) to the promoter suggests a path for promoter DNA relative to the RNA polymerase surface in this archaeal transcription initiation complex. In addition, an unidentified protein approximately the size of TBP cross-links to the non-transcribed DNA strand near the upstream edge of the transcription bubble. Cross-linking is specific to the polymerase-containing initiation complex and requires the gdh promoter TATA box. The location of this protein suggests that it, like TFB1, could also have a role in transcription initiation following RNA polymerase recruitment.

A common site on TBP for transcription by RNA polymerases II and III.

The TATA-binding protein (TBP) is involved in all nuclear transcription. We show that a common site on TBP is used for transcription initiation complex formation by RNA polymerases (pols) II and III. TBP, the transcription factor IIB (TFIIB)-related factor Brf1 and the pol III-specific factor Bdp1 constitute TFIIIB. A photochemical cross-linking approach was used to survey a collection of human TBP surface residue mutants for their ability to form TFIIIB-DNA complexes reliant on only the TFIIB-related part of Brf1. Mutations impairing complex formation and transcription were identified and mapped on the surface of TBP. The most severe effects were observed for mutations in the C-terminal stirrup of TBP, which is the principal site of interaction between TBP and TFIIB. Structural modeling of the Brf1-TBP complex and comparison with its TFIIB-TBP analog further rationalizes the close resemblance of the TBP interaction with the N-proximal part of Brf1 and TFIIB, and establishes the conserved usage of a TBP surface in pol II and pol III transcription for a conserved function in the initiation of transcription.