A holistic view of the complex Recent studies on gene regulation in Saccharomyces cerevisiae support the view that eukaryotic activators stimulate transcription by recruiting an RNA polymerase I.holoenzyme to the promoter in a single step. (1995)

A textbook combination of biochemistry and geneticshas suggested a new model for gene activation in theyeast Saccharomyces cerevisiae. The model is based on thepremise that the transcriptional apparatus in yeast existsin the form of a holoenzyme containing both RNApolymerase II and ancillary factors, which endow it withthe capacity to recognize promoter DNA and respond toupstream activators. In this scheme, an activator wouldstimulate transcription by contacting a surface of theholoenzyme and recruiting it to DNA in a single step -a mechanism conceptually similar to gene activation inprokaryotic cells. The holoenzyme model arose fromtwo observations: the biochemical isolation of a largelypreassembled transcription complex from yeast extracts[1,2] and experiments in vivo, which indicated that teth-ering diverse components of the yeast transcriptionalmachinery to DNA could circumvent the requirementfor upstream activators [3-5].Although the concept of a holoenzyme originated fromstudies of the bacterium Escherichia coli over two decadesago, its existence in eukaryotic cells was unexpected. Thetraditional view of eukaryotic gene activation proposedthat activators stimulate the stepwise assembly of a multi-component transcription complex, comprising RNApolymerase II and six general factors called TFIIA,TFIIB, TFIID, TFIIE, TFIIF and TFIIH, which are con-served between yeast and humans [6]. The assembly ofthe complex was thought to be nucleated by the bindingof TFIID to the TATA box, and to culminate in thebinding of TFIIH. This notion predominated untilindependent discoveries by the laboratories of RogerKornberg and Rick Young of a form of RNA poly-merase II that was associated with proteins not consid-ered part of the core enzyme [1,2]. Both laboratoriesreferred to these macromolecular assemblies as holo-enzymes, although the term is not strictly correct bycomparison to the prokaryotic prototype.The holoenzyme isolated by Young and co-workersemerged from a genetic analysis of the carboxy-terminaldomain of the largest RNA polymerase II subunit(reviewed in [7]). This domain contains 26 heptapeptiderepeats, the phosphorylation of which by cyclin-depen-dent kinases apparently plays a role in transcriptionalcontrol. Deletion of all but 10-12 of these repeats con-fers a cold-sensitive phenotype on yeast. Genetic sup-pressors of the conditional lethal phenotype, termedsuppressors of RNA polymerase B (II) or SRBs, restorethe ability of the yeast to grow in the cold. Although theprecise physiological functions of the SRB proteins arenot fully understood, biochemical analysis reveals thatthey are associated with a complex containing stoichio-metric amounts of RNA polymerase II and the yeastgeneral transcription factors TFIIB, TFIIH and TFIIF[2]. This complex can support both basal and activator-responsive transcription in vitro upon addition of onlyTFIIE and the TATA-box-binding protein (TBP), a sub-unit of TFIID. Kornberg and colleagues isolated a smallerversion of the holoenzyme, comprising RNA poly-merase II and a complex of proteins termed the mediator[1]. Analysis of the mediator showed it to be composedof the SRBs, TFIIF and two co-activators called SUG1and GAL11, later found to be components of theholoenzyme isolated by Young.The observation by Young and colleagues that only afraction of the cellular RNA polymerase II and generaltranscription factors is present in the holoenzyme initiallyraised doubts about its authenticity. However, becausealmost all of the SRBs are associated, the finding that atemperature-sensitive SRB4 mutant leads to a completeshut-off of mRNA synthesis at the restrictive tempera-ture [8] provided a compelling argument that theholoenzyme is the transcriptionally active form of RNApolymerase in vivo. Such a discovery has broad implica-tions for the mechanism of eukaryotic gene activation,perhaps best considered by using the E. coli holoenzymeas a model (Fig. la,b).The E. coli RNA polymerase holoenzyme consists of afour-subunit core (a