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.

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.

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.

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.

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.

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.

Essential roles of Bdp1, a subunit of RNA polymerase III initiation factor TFIIIB, in transcription and tRNA processing.

The essential Saccharomyces cerevisiae gene BDP1 encodes a subunit of RNA polymerase III (Pol III) transcription factor (TFIIIB); TATA box binding protein (TBP) and Brf1 are the other subunits of this three-protein complex. Deletion analysis defined three segments of Bdp1 that are essential for viability. A central segment, comprising amino acids 327 to 353, was found to be dispensable, and cells making Bdp1 that was split within this segment, at amino acid 352, are viable. Suppression of bdp1 conditional viability by overexpressing SPT15 and BRF1 identified functional interactions of specific Bdp1 segments with TBP and Brf1, respectively. A Bdp1 deletion near essential segment I was synthetically lethal with overexpression of PCF1-1, a dominant gain-of-function mutation in the second tetracopeptide repeat motif (out of 11) of the Tfc4 (tau(131)) subunit of TFIIIC. The analysis also identifies a connection between Bdp1 and posttranscriptional processing of Pol III transcripts. Yeast genomic library screening identified RPR1 as the specific overexpression suppressor of very slow growth at 37 degrees C due to deletion of Bdp1 amino acids 253 to 269. RPR1 RNA, a Pol III transcript, is the RNA subunit of RNase P, which trims pre-tRNA transcript 5' ends. Maturation of tRNA was found to be aberrant in bdp1-Delta 253-269 cells, and RPR1 transcription with the highly resolved Pol III transcription system in vitro was also diminished when recombinant Bdp1 Delta 253-269 replaced wild-type Bdp1. Physical interaction of RNase P with Bdp1 was demonstrated by coimmunoprecipitation and pull-down assays.

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.

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.

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.

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.

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.

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.

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.

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.

Thermoirreversible and thermoreversible promoter opening by two Escherichia coli RNA polymerase holoenzymes.

Promoter opening, in which the complementary DNA strands separate around the transcriptional start site, is generally thermoreversible. An exceptional case of thermoirreversible opening of the T4 late promoter has been analyzed by KMnO4 footprinting and transcription. T4 late promoters, which consist of an 8-base pair (bp) TATA box "-10" element, are recognized by the small, phage-encoded, highly diverged sigma-family initiation subunit gp55. The T4 late promoter only opens above 15-20 degrees C, but once it has been formed remains open and transcriptionally active for days at -0.5 degrees C. The low temperature-trapped open complex and its isothermally formed state are shown to be structurally distinctive. Two "extended -10" sigma 70 promoters, which, like the T4 late promoter, lack "-35" sites, have been subjected to a comparative analysis: the T4 middle promoter PrIIB2 opens and closes thermoreversibly under conditions of basal and MotA- and AsiA-activated transcription. The open galP1 promoter complex, whose transcription bubble is very AT-rich, also closes reversibly upon shift to -0.5 degrees C, but more slowly than does the rIIB2 promoter. Formation of a trapped-open low temperature state of the promoter complex appears to be a singular property of gp55-RNA polymerase holoenzyme.

Effects of DNA strand breaks on transcription by RNA polymerase III: insights into the role of TFIIIB and the polarity of promoter opening.

Certain deletion mutants of the Brf1 and Bdp1 subunits of transcription factor (TF) IIIB retain the ability to recruit RNA polymerase (pol) III to its promoters, but fail to support promoter opening: deletions within an internal Bdp1 segment interfere with initiation of DNA strand separation, and an N-terminal Brf1 deletion blocks propagation of promoter opening past the transcriptional start site. The ability of DNA strand breaks to restore pol III transcription activity to these defective TFIIIB assemblies has been analyzed using U6 snRNA gene constructs. Breaks in a 21 bp segment spanning the transcriptional start rescue transcription in DNA strand-specific and subunit/mutation-specific patterns. A cluster of Bdp1 internal deletions also reverses the inactivation of transcription with wild-type TFIIIB generated by certain transcribed (template) strand breaks near the transcriptional start site. A structure-based model and topological considerations interpret these observations, explain how Bdp1 and Brf1 help to enforce the general upstream--> downstream polarity of promoter opening and specify requirements for polarity reversal.