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NC2 (Dr1-Drap1 or Bur6-Ydr1) has been characterized in vitro as a general negative regulator of RNA polymerase II (Pol II) transcription that interacts with TATA-binding protein (TBP) and inhibits its function. Here, we show that NC2 associates with promoters in vivo in a manner that correlates with transcriptional activity and with occupancy by basal transcription factors. NC2 rapidly associates with promoters in response to transcriptional activation, and it remains associated under conditions in which transcription is blocked after assembly of the Pol II preinitiation complex. NC2 positively and negatively affects approximately 17% of Saccharomyces cerevisiae genes in a pattern that resembles the response to general environmental stress. Relative to TBP, NC2 occupancy is high at promoters where NC2 is positively required for normal levels of transcription. Thus, NC2 is associated with the Pol II preinitiation complex, and it can play a direct and positive role at certain promoters in vivo.

The carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II is phosphorylated soon after transcriptional initiation. We show here that the essential FCP1 gene of S. cerevisiae is linked genetically to RNA polymerase II and encodes a CTD phosphatase essential for dephosphorylation of RNA polymerase II in vivo. Fcp1p contains a phosphatase motif, psi psi psi DXDX(T/V)psi psi, which is novel for eukaryotic protein phosphatases and essential for Fcp1p to function in vivo. This motif is also required for recombinant Fcp1p to dephosphorylate the RNA polymerase II CTD or the artificial substrate p-nitrophenylphosphate in vitro. The effects of fcp1 mutations in global run-on and genome-wide expression studies show that transcription by RNA polymerase II in S. cerevisiae generally requires CTD phosphatase.

Genome-wide expression analysis was used to identify genes whose expression depends on the functions of key components of the transcription initiation machinery in yeast. Components of the RNA polymerase II holoenzyme, the general transcription factor TFIID, and the SAGA chromatin modification complex were found to have roles in expression of distinct sets of genes. The results reveal an unanticipated level of regulation which is superimposed on that due to gene-specific transcription factors, a novel mechanism for coordinate regulation of specific sets of genes when cells encounter limiting nutrients, and evidence that the ultimate targets of signal transduction pathways can be identified within the initiation apparatus.

We have analyzed transcription initiation by RNA polymerase II (pol II) in a highly efficient in vitro transcription system composed of essentially homogeneous protein preparations. The pol II complex was stalled on adenovirus major late promoter templates at defined positions, and the open region and RNA products of these complexes were examined. The first transition is formation of the open complex, which can be reversed by addition of ATPgammaS. The open region is no longer sensitive to ATPgammaS after formation of a four-nucleotide RNA, which constitutes the second transition. This indicates that the ATP-dependent DNA helicase activity of TFIIH is required to maintain the open region only during formation of the first three phosphodiester bonds. The downstream part of the transcription bubble expands in a continuous motion, but the initially opened region (-9/-2 on the non-template strand) recloses abruptly when transcription reaches register 11. This third transition is accompanied by a switch from abortive to productive RNA synthesis, which implies promoter clearance. Our findings provide a framework to analyze regulation of these specific transitions during transcription initiation by pol II.

Open complex formation precedes initiation of transcription by RNA polymerases. In the analysis of transcription initiation from eukaryotic class II promoters, we have used promoter DNA structures that represent intermediates in open complex formation. We describe the preparation and isolation of heteroduplex promoter fragments. Probes containing these DNA structures have a general application in the study of proteins binding to junctions of double- and single-stranded DNA. Such proteins play important roles not only in the regulation of RNA synthesis but also in processes like repair, replication, and recombination of DNA. In addition, a protocol is provided for a rapid and quantitative assay for open complexes and transcription bubbles using potassium permanganate as a chemical probe for single-stranded regions in DNA.

We have studied promoter opening in assays reconstituted with purified RNA polymerase II and basal transcription factors. We found that creating a region of heteroduplex DNA around the start site of the adenovirus major late (AdML) promoter circumvents the requirement for TFIIE and TFIIH in transcription. The critical size and position of the heteroduplex region that alleviates the requirement for TFIIE and TFIIH is six nucleotides, from -4 to +2. Promoter opening was investigated directly with potassium permanganate (KMnO4), a chemical probe specific for single-stranded thymidines. We found that KMnO4-detectable opening of the AdML promoter requires the presence of the complete pre-initiation complex, DBpolFEH, and that opening occurs in two discrete steps. First, dependent on ATP but prior to initiation, the -9 to +1 region becomes single-stranded. Second, formation of the first phosphodiester bond results in expansion of the open region to the +8 position. Our results lead to a model in which the critical function of the TFIIH-associated DNA helicases is to create a single-stranded region. This gives RNA polymerase II access to the nucleotides of the template strand and allows expansion of the open region upon formation of the first phosphodiester bond.