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Although we have made significant progress, we still possess a limited understanding of how genomic and epigenomic information directs gene expression programs through sequence-specific transcription factors (TFs). Extensive research has settled on three general classes of TF targets in metazoans: promoter accessibility via chromatin regulation (e.g., SAGA), assembly of the general transcription factors on promoter DNA (e.g., TFIID), and recruitment of RNA polymerase (Pol) II (e.g., Mediator) to establish a transcription pre-initiation complex (PIC). Here we discuss TFs and their targets. We also place this in the context of our current work with Saccharomyces (yeast), where we find that promoters typically lack an architecture that supports TF function. Moreover, yeast promoters that support TF binding also display interactions with cofactors like SAGA and Mediator, but not TFIID. It is unknown to what extent all genes in metazoans require TFs and their cofactors.

The heterodimeric transcription factor E2F1-DP1 plays crucial roles in coordinating gene expression during G1/S cell cycle progression. For transcriptional activation, the transactivation domain (TAD) of E2F1 is known to interact with the TATA-binding protein of TFIID and the p62 subunit of TFIIH. It is generally believed that DP1 facilitates E2F1 binding to target DNA and does not possess a TAD. Here, we show that an acidic region of DP1, whose function has remained elusive, binds to the plekstrin homology (PH) domain of p62 with higher affinity than that of E2F1 and contributes to transcriptional activation. The structure of the complex revealed that DP1 forms a twisted U-shaped, string-like conformation and binds to the surface of the PH domain by anchoring Phe403 into a pocket in the PH domain. The transcriptional activity of E2F1-DP1 was reduced when Phe403 of DP1 was mutated. These findings indicate that the acidic region of DP1 acts as a TAD by contacting TFIIH.

Pre-mRNA maturation frequently occurs at the same time and place as transcription by RNA polymerase II (pol II). The co-transcriptionality of mRNA processing has permitted the evolution of mechanisms that functionally couple transcription elongation with diverse events that occur on the nascent RNA. This review summarizes current understanding of the relationship between transcriptional elongation through a chromatin template and co-transcriptional splicing including alternative splicing decisions that affect the expression of most human genes.

The B2 family of short interspersed elements is transcribed into non-coding RNA by RNA polymerase III. The ~180-nt B2 RNA has been shown to potently repress mRNA transcription by binding tightly to RNA polymerase II (Pol II) and assembling with it into complexes on promoter DNA, where it keeps the polymerase from properly engaging the promoter DNA. Mammalian Pol II is an ~500-kDa complex that contains 12 different protein subunits, providing many possible surfaces for interaction with B2 RNA. We found that the carboxy-terminal domain of the largest Pol II subunit was not required for B2 RNA to bind Pol II and repress transcription in vitro. To identify the surface on Pol II to which the minimal functional region of B2 RNA binds, we coupled multi-step affinity purification, reversible formaldehyde cross-linking, peptide sequencing by mass spectrometry, and analysis of peptide enrichment. The Pol II peptides most highly recovered after cross-linking to B2 RNA mapped to the DNA binding cleft and active-site region of Pol II. These studies determine the location of a defined nucleic acid binding site on a large, native, multi-subunit complex and provide insight into the mechanism of transcriptional repression by B2 RNA.

In eukaryotes, RNA polymerase II requires general transcription factors to initiate mRNA transcription. TFIIE subunits α and β form a heterodimer and recruit TFIIH to complete the assembly of the pre-initiation complex. Here, we have determined the crystal structure of human TFIIE at atomic resolution. The N-terminal half of TFIIEα forms an extended winged helix (WH) domain with an additional helix, followed by a zinc-finger domain. TFIIEβ contains the WH2 domain, followed by two coiled-coil helices intertwining with TFIIEα. We also showed that TFIIEα binds to TFIIEβ with nanomolar affinity using isothermal titration calorimetry. In addition, mutations on the residues involved in the interactions resulted in severe growth defects in yeast. Lack of the C-terminal region of yeast TFIIEβ causes a mild growth defect in vivo. These findings provide a structural basis for understanding the functional mechanisms of TFIIE in the context of pre-initiation complex formation and transcription initiation.

The positive transcription elongation factor b (P-TEFb), composed of cyclin-dependent kinase 9 and cyclin T1, stimulates the elongation of transcription by hyperphosphorylating the C-terminal region of RNA polymerase II. Aberrant activation of P-TEFb results in manifestations of cardiac hypertrophy in mice, suggesting that P-TEFb is an essential factor for cardiac myocyte function and development. Here, we present evidence that P-TEFb selectively activates transcription mediated by the myocyte enhancer factor 2 (MEF2) family of transcription factors, key regulatory factors for myocyte development. Knockdown of endogenous cyclin T1 in murine C2C12 cells abolishes MEF2-dependent reporter gene expression as well as transcription of endogenous MEF2 target genes, whereas overexpression of P-TEFb enhances MEF2-dependent transcription. P-TEFb interacts with MEF2 both in vitro and in vivo. Activation of MEF2-dependent transcription induced by serum starvation is mediated by a rapid dissociation of P-TEFb from its inhibitory subunit, HEXIM1, and a subsequent recruitment of P-TEFb to MEF2 binding sites in the promoter region of MEF2 target genes. These results indicate that recruitment of P-TEFb is a critical step for stimulation of MEF2-dependent transcription, therefore providing a fundamentally important regulatory mechanism underlying the transcriptional program in muscle cells.

Nhp6p is an architectural Saccharomyces cerevisiae non-histone chromosomal protein that bends DNA and plays an important role in transcription and genome stability. We used the split-ubiquitin system to isolate proteins that interact with Nhp6p in vivo, and we confirmed 11 of these protein-protein interactions with glutathione S-transferase pull-down experiments in vitro. Most of the Nhp6p-interacting proteins are involved in transcription and DNA repair. We utilized the ZDS1, PUR5 and UME6 genes, which are repressed by Nhp6p and its interacting partners Rpb4p and Med3p, to study the chromosomal localization of these three proteins in wild-type and gene deletion strains. Nhp6p, Med3p and Rpb4p were found at the promoters of ZDS1, PUR5 and UME6, indicating that the repressing effects the three proteins had on the expression of these three genes had been direct ones. We also found that Med3p inhibited promoter clearance of RNA polymerase II, which contained the dissociable subunit Rpb4p, while Nhp6p recruited Rpb4p to the basal promoters of ZDS1, PUR5 and UME6. Our results further suggest that Rpb4p inhibits transcription initiation but stimulates transcription elongation and that Nhp6p and Med3p regulate gene expression by controlling the local subunit composition of RNA polymerase II.

Related multisubunit RNA polymerases (RNAPs) carry out gene transcription in all kingdoms of life. Since structural information is limited to bacterial and eukaryotic RNAPs, we determined the cryo-electron microscopic structure of the RNAP from the thermophilic archaeon Pyrococcus furiosus at 13 A resolution. Comparison with eukaryotic RNAP II reveals a conserved architecture, no homologues for subunits Rpb8 and Rpb9, and significant deviation in the polymerase foot, jaws, pore, and protrusion. The structural organization of the archaeal RNA polymerase serves as a reference for future structure-function analysis of the transcription mechanism and allows for evolutionary comparisons.

During transcription initiation conformational changes in the transcriptional machinery are required to accommodate the growing RNA, to allow the polymerase to release the promoter, and to endow the elongation complex with high processivity. In T7 RNA polymerase these changes involve refolding and reorientation of elements of the N-terminal domain, as well as changes in how the DNA is bound within the complex. However, when and where these conformational changes occur is unknown, and the role of these changes in allowing the polymerase to disengage the promoter is poorly understood. To address this we have used chemical nucleases tethered to the polymerase to monitor conformational changes, and engineered disulfide bonds to block conformational changes at defined steps in transcription. We find that many of the major structural transitions occur cooperatively, at a point coincident with promoter release. Moreover, promoter release requires that two elements of the polymerase which form a continuous promoter recognition surface in the initial transcription complex move apart: if this movement is blocked the polymerase cannot disengage the promoter.

Many activators of transcription are sequence-specific DNA-binding proteins that stimulate transcription initiation through interaction with RNA polymerase (RNAP). Such activators can be constructed artificially by fusing a DNA-binding protein to a protein domain that can interact with an accessible surface of RNAP. In these cases, the artificial activator is directed to a target promoter bearing a recognition site for the DNA-binding protein. Here we describe an artificial activator that functions by contacting a normally occluded surface of promoter-bound RNAP holoenzyme. This artificial activator consists of a DNA-binding protein fused to the bacteriophage T4-encoded transcription regulator AsiA. On its own, AsiA inhibits transcription by Escherichia coli RNAP because it remodels the holoenzyme, disrupting an intersubunit interaction that is required for recognition of the major class of bacterial promoters. However, when tethered to the DNA via a DNA-binding protein, AsiA can exert a strong stimulatory effect on transcription by disrupting the same intersubunit interaction, contacting an otherwise occluded surface of the holoenzyme. We show that mutations that affect the intersubunit interaction targeted by AsiA modulate the stimulatory effect of this artificial activator. Our results thus demonstrate that changes in the accessibility of a normally occluded surface of the RNAP holoenzyme can modulate the activity of a gene-specific regulator of transcription.

Transcription regulation relies in the molecular interplay between the RNA polymerase (RNAP) and regulatory factors. Phage φ29 promoters A2c, A2b and A3 are coordinately regulated by the transcriptional regulator protein p4 and the histone-like protein p6. This study shows that protein p4 binds simultaneously to four sites: sites 1 and 2 located between promoters A2c and A2b and sites 3 and 4 between promoters A2b and A3, placed in such a way that bound p4 is equidistant from promoters A2c and A2b and one helix turn further upstream from promoter A3. The p4 molecules bound to sites 1 and 3 reorganise the binding of protein p6, giving rise to the nucleoprotein complex responsible for the switch from early to late transcription. We identify the positioning of the αCTD-RNAP domain at these promoters, and demonstrate that the domains are crucial for promoter A2b recognition and required for full activity of promoter A2c. Since binding of RNAP overlaps with p4 and p6 binding, repression of the early transcription relies on the synergy of the regulators able to antagonize the stable binding of the RNAP through competition for the same target, while activation of late transcription is carried out through the stabilization of the RNAP by the p4/p6 nucleoprotein complex. The control of promoters A2c and A2b by feed-back regulation is discussed., This work was funded by grants 2 R01 GM27242-24 from the National Institutes of Health and BMC 2002-03818 from the Spanish Ministry of Science and Technology and by an Institutional grant from Fundación Ramón Areces to the Centro de Biologı́a Molecular “Severo Ochoa”. The “Ramón y Cajal” program of the Spanish Ministry of Science and Technology supported A.C.

RNA polymerase II-dependent transcription requires the assembly of a multi-protein, preinitiation complex on core promoter elements. Transcription factor IID (TFIID) comprising the TATA box-binding protein (TBP) and TBP-associated factors (TAFs) is responsible for promoter recognition in this complex. Subsequent association of TFIIA and TFIIB provides enhanced complex stability. TFIIA is required for transcriptional stimulation by certain viral and cellular activators, and favors formation of the preinitiation complex in the presence of repressor NC2. The X-ray structures of human and yeast TBP/TFIIA/DNA complexes at 2.1A and 1.9A resolution, respectively, are presented here and seen to resemble each other closely. The interactions made by human TFIIA with TBP and DNA within and upstream of the TATA box, including those involving water molecules, are described and compared to the yeast structure. Of particular interest is a previously unobserved region of TFIIA that extends the binding interface with TBP in the yeast, but not in the human complex, and that further elucidates biochemical and genetic results.

Transcriptional activation of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) promoter element is regulated by the essential viral Tat protein that binds to the viral TAR RNA target and recruits a positive transcription elongation complex (P-TEFb). We have used a stepwise transcription approach and a highly sensitive assay to determine the dynamics of interactions between HIV-1 Tat and the transcription complexes actively engaged in elongation. Our results demonstrate that Tat protein associates with RNA polymerase II complexes during early transcription elongation after the promoter clearance and before the synthesis of full-length TAR RNA transcript. This interaction of Tat with RNA polymerase II elongation complexes is P-TEFb-independent. Our results also show that there are two Tat binding sites on each transcription elongation complex; one is located on TAR RNA and the other one on RNA polymerase II near the exit site for nascent mRNA transcripts. These findings suggest that two Tat molecules are involved in performing various functions during a single round of HIV-1 mRNA synthesis.

We have used the fission yeast Schizosaccharomyces pombe to establish both a biochemical and genetic system to study the roles of general transcription factors in transcription initiation. Extracts were prepared that faithfully transcribed S. pombe promoters and the results confirm that, in contrast to the budding yeast Saccharomyces cerevisiae, in vitro transcription in S. pombe initiates near to the TATA element. S. pombe transcription relies on upstream activation sequence elements and these can be replaced successfully with sites for binding Gal4-VP16 activators. Although it is mammalian-like in these respects, S. pombe initiation uses an unusual scanning mechanism. This directs initiation, preferentially using purines, within a narrow window approximately 25-40 base-pairs downstream from the edge of the TATA element. Genetic experiments showed that this scanning mechanism was associated with the properties of the TFIIB polypeptide. When human TFIIB was expressed in S. pombe, it was accepted by the endogenous transcription machinery and caused initiation to be restricted to the closer edge of this window, corresponding to the distance in humans. Preliminary experiments suggested that S. cerevisiae TFIIB was not accepted. The results enlarge the potential for using fission yeast to study the properties of general transcription factors such as TFIIB in choosing the sites at which transcription initiates.

The CCR4-NOT complex is an evolutionarily conserved, transcriptional regulatory complex that is involved in controlling mRNA initiation, elongation and degradation. The CCR4-NOT proteins from Saccharomyces cerevisiae exist in two complexes, 1.9×106 Da and 1.0×106 Da (1.0 MDa) in size, and individual components of these complexes display such disparate functions as binding to and restricting TFIID functions, contacting SAGA and contributing to mRNA deadenylation. As a first step in characterizing the functional roles of the 1.0 MDa complex, we have purified it to near homogeneity. Mass spectrometric analysis was subsequently used to identify all the components of the complex. The 1.0 MDa complex was found to contain CCR4, CAF1, NOT1-5 and two new proteins, CAF40 and CAF130. CAF130 and CAF40 are two unique yeast proteins, with CAF40 displaying extensive homology to proteins from other eukaryotes. Immunoprecipitation and gel filtration experiments confirmed that CAF130 and CAF40 are components of both of the 1.9 MDa and 1.0 MDa CCR4-NOT complexes. Biochemical analysis indicated that the CAF40 and CAF130 proteins bind to the NOT1 protein and exist in a location separate from the two other subsets of proteins in the complex: the CCR4 and CAF1 proteins, and the NOT2, NOT4 and NOT5 proteins. Moreover, CAF40 was able to interact with human NOT1, suggesting that human CAF40 would also be a component of the recently identified human CCR4-NOT complex. Analysis of caf40 and caf130 deletions indicated that they elicited phenotypes shared by defects in other CCR4-NOT genes. The distinct location of CAF40 and CAF130 and the evolutionary conservation of CAF40 implicate them in novel roles in the function of the CCR4-NOT complex.

Interleukin-2 (IL-2) is a cytokine critical for the proper stimulation of T-cells during the mammalian immune response. Shortly after T-cell stimulation, transcription of the IL-2 gene is upregulated. Here, we studied the kinetic mechanism of basal transcription at the IL-2 promoter using a human in vitro RNA polymerase II transcription system. We experimentally divided the transcription reaction into discrete steps, including preinitiation complex formation, initiation, escape commitment, and promoter escape. Using pre-steady state approaches, we measured the rate at which each of these steps occurs. We found that the rate of functional preinitiation complex formation limits the overall rate of transcription at the IL-2 promoter under the conditions described here. Furthermore, we found that the recruitment of TFIIF and RNA polymerase II to a TFIID/TFIIA/TFIIB/promoter complex dictates the rate of preinitiation complex formation. The rate of synthesis of 28 nt RNA from preinitiation complexes was rapid compared to the rate of preinitiation complex formation. Moreover, we found that the synthesis of a four nucleotide RNA was necessary and sufficient to rapidly complete the escape commitment step of transcription at the IL-2 promoter. Comparative experiments with the adenovirus major late promoter revealed that, while the overall mechanism of transcription is the same at the two promoters, promoter sequence and/or architecture dictate the rate of promoter escape. We present a kinetic model for a single round of basal transcription at the IL-2 promoter that provides insight into mechanisms by which the IL-2 gene is transcriptionally regulated.

International audience; Formaldehyde cross-linking was used in a kinetic analysis of RNA polymerase-lacUV5 promoter interactions in open complexes (RP(o)). RP(o) quenched from 37 degrees C to 14 degrees C isomerised to a closed, competitor resistant, complex (RP(LT)). We observed that contacts of the beta' and sigma subunits with the positions -3, -5 of the non-template DNA strand disappeared very quickly during the first 30 seconds after the temperature downshift. The re-annealing of the DNA downstream of the transcription start site takes place in the same time scale. However re-annealing of the upstream part of the transcription bubble was slower and completed within five minutes. The results support a two-step model of promoter melting and suggest that conformational changes in the RNA polymerase occur concurrently with the melting around the transcription start site.

Bacterial RNA polymerase and eukaryotic RNA polymerase II exhibit striking structural similarities, including similarities in overall structure, relative positions of subunits, relative positions of functional determinants, and structures and folding topologies of subunits. These structural similarities are paralleled by similarities in mechanisms of interaction with DNA.

General transcription factor IIF (TFIIF) is required for transcription by RNA polymerase II; it consists minimally of a heterodimer of RNA polymerase-associated proteins RAP30 and RAP74. According to solution and mutagenesis studies, the multiple domains of RAP30 and RAP74 bind PolII, TFIIB, TAF250 and DNA in interactions that are essential for transcription initiation and elongation. The X-ray structure of the RAP30/RAP74 interaction domains at 1.7 A resolution reveals a novel "triple barrel" dimerization fold and suggests with mutant data that interactions with the transcription apparatus are mediated not only by this tripartite beta-barrel, but also via flexible loops and alpha and beta-structures extending from it.

SINEs, short interspersed repeated DNA elements, undergo amplification through retroposition and subsequent integration into a new location in the genome. Each new SINE insertion will be located in a new chromosomal environment, with different flanking sequences. Modulation of transcription by different flanking sequences may play an important role in determining which SINE elements are preferentially active in a genome. We evaluated the ability of upstream flanking sequences to regulate the transcription of three different SINEs (Alu, B2 and ID) by constructing chimeric constructs with known 5′ flanking sequences of RNA polymerase III-transcribed genes. Upstream sequences from the 7SL RNA gene, U6 RNA gene, vault RNA gene, and BC1 gene increase transcription of Alu, B2 and BC1 in transient transfections of NIH3T3, HeLa, Neuro2a and C6 glioma cell lines. The 7SL sequence proved most efficient in increasing SINE transcription. The 7SL upstream fused to the BC1 RNA gene (an ID element) was used to create a transgenic mouse line. In contrast to the tissue-specific endogenous BC1 transcription, BC1 transgene transcripts were detected in all tissues tested. However, expression was much higher in those tissues that express the endogenous gene, demonstrating both transcriptional and post-transcriptional regulation. The BC1 RNA was detected in a similar ribonucleoprotein complex in the different tissues.

The most peculiar transcriptional property of eukaryotic tRNA genes, as well as of other genes served by RNA polymerase III, is their complete dependence on the intragenic interaction platform provided by transcription factor IIIC (TFIIIC) for the productive assembly of the TBP-containing initiation factor TFIIIB. The sole exception, in yeast, is the U6 RNA gene, which is able to exploit a TATAAATA element, 30 bp upstream of the transcription start site, for the TFIIIC-independent assembly of TFIIIB. To find out whether this extragenic core promoter organization and autonomous TFIIIB assembly capacity are unique features of the U6 gene or also apply to other genes transcribed by RNA polymerase III, we scanned the 5′-flanking regions (up to position −100) of the entire tRNA gene set of Saccharomyces cerevisiae searching for U6-like TATA motifs. Four tRNA genes harboring such a sequence motif around position −30 were identified and found to be transcribed in vitro by a minimal system only composed of TFIIIB and RNA polymerase III. In this system, start site selection is not at all affected by the absence of TFIIIC, which, when added, significantly stimulates transcription by determining an increase in the number, rather than in the efficiency of utilization, of productive initiation complexes. A specific TBP-TATA element interaction is absolutely required for TFIIIC-independent transcription, but the nearby sequence context also contributes to the efficiency of autonomous TFIIIB assembly. The existence of a TFIIIB assembly pathway leading to the faithful transcription of natural eukaryotic tRNA genes in the absence of TFIIIC provides novel insights into the functional flexibility of the eukaryotic tRNA gene transcription machinery and on its evolution from an ancestral RNA polymerase III system relying on upstream, TATA-centered control elements.

Ribosomal RNA transcription initiation requires the melting of DNA to form an open complex, formation of the first few phosphodiester bonds, commencement of RNA polymerase I movement along the DNA, clearance of the promoter, and the formation of a steady-state ternary elongation complex. We examined DNA melting and promoter clearance by using potassium permanganate, diethylpyrocarbonate and methidiumpropylEDTA.Fe(II) footprinting. In combination, these methods demonstrated: (1) TIF-IB and RNA polymerase I are the only proteins required for formation of an initial approximately 9 base-pair open promoter region. This finding contradicts earlier results using diethylpyrocarbonate alone, which suggested an RNA synthesis requirement for stable melting. (2) DNA melting is temperature-dependent, with a tm between 15 and 20 degrees C. (3) Temperature-dependency of melting, as well as stalling the polymerase at sites close to the transcription start site revealed that the melted DNA region initially opens upstream of the transcription initiation site, and enlarges in a downstream direction coordinate with initiation, eventually attaining a steady-state transcription bubble of approximately 19 base-pairs. (4) The RNA-DNA hybrid protects the template DNA from single-strand footprinting reagents. The hybrid is 9 bp in length, consistent with the longer hybrid estimated by some for the Escherichia coli polymerase and with the hybrids estimated for eukaryotic polymerases II and III.

The complementary DNAs of the 12 subunits of fission yeast (Schizosaccharomyces pombe) RNA polymerase II were expressed from strong promoters in Saccharomyces cerevisiae and tested for heterospecific complementation by monitoring their ability to replace in vivo the null mutants of the corresponding host genes. Rpb1 and Rpb2, the two largest subunits and Rpb8, a small subunit shared by all three polymerases, failed to support growth in S. cerevisiae. The remaining nine subunits were all proficient for heterospecific complementation and led in most cases to a wild-type level of growth. The two alpha-like subunits (Rpb3 and Rpb11), however, did not support growth at high (37 degrees C) or low (25 degrees C) temperatures. In the case of Rpb3, growth was restored by increasing the gene dosage of the host Rpb11 or Rpb10 subunits, confirming previous evidence of a close genetic interaction between these three subunits.

Phage T7 RNA polymerase is the only DNA-dependent RNA polymerase for which we have a high-resolution structure of the promoter-bound complex. Recent studies with the more complex RNA polymerases have suggested a role for DNA wrapping in the initiation of transcription. Here, circular permutation gel retardation assays provide evidence that the polymerase does indeed bend its promoter DNA. A complementary set of experiments employing differential phasing from an array of phased A-tracts provides further evidence for both intrinsic and polymerase-induced bends in the T7 RNA polymerase promoter DNA. The bend in the complex is predicted to be about 40-60 degrees and to be centered around positions -2 to +1, at the start site for transcription, while the intrinsic bend is much smaller (about 10 degrees ). These results, viewed in the light of a recent crystal structure for the complex, suggest a mechanism by which binding leads directly to bending. Bending at the start site would then facilitate the melting necessary to initiate transcription.

Transcription activation by Escherichia coli catabolite activator protein (CAP) at each of two classes of simple CAP-dependent promoters is understood in structural and mechanistic detail. At class I CAP-dependent promoters, CAP activates transcription from a DNA site located upstream of the DNA site for RNA polymerase holoenzyme (RNAP); at these promoters, transcription activation involves protein-protein interactions between CAP and the RNAP alpha subunit C-terminal domain that facilitate binding of RNAP to promoter DNA to form the RNAP-promoter closed complex. At class II CAP-dependent promoters, CAP activates transcription from a DNA site that overlaps the DNA site for RNAP; at these promoters, transcription activation involves both: (i) protein-protein interactions between CAP and RNAP alpha subunit C-terminal domain that facilitate binding of RNAP to promoter DNA to form the RNAP-promoter closed complex; and (ii) protein-protein interactions between CAP and RNAP alpha subunit N-terminal domain that facilitates isomerization of the RNAP-promoter closed complex to the RNAP-promoter open complex. Straightforward combination of the mechanisms for transcription activation at class I and class II CAP-dependent promoters permits synergistic transcription activation by multiple molecules of CAP, or by CAP and other activators. Interference with determinants of CAP or RNAP involved in transcription activation at class I and class II CAP-dependent promoters permits "anti-activation" by negative regulators. Basic features of transcription activation at class I and class II CAP-dependent promoters appear to be generalizable to other activators.

The human immunodeficiency? virus type-1 (HIV-1) Tat protein regulates transcription by stimulating RNA polymerase processivity. Using immobilised templates, we have been able to study the effects of Tat on protein kinase activity during the pre-initiation and elongation stages of HIV-1 transcription. In pre-initiation complexes formed at the HIV-1 LTR, the C-terminal domain (CTD) of RNA polymerase II is rapidly phosphorylated by transcription factor IIH (TFIIH). Addition of Tat does not affect either the rate or the extent of CTD phosphorylation in the pre-initiation complexes. By contrast, Tat is able to stimulate additional CTD phosphorylation in elongation complexes. This reaction creates a novel form of the RNA polymerase that we have called RNA polymerase IIo∗. Formation of the RNA polymerase IIo∗ occurs only after transcription of templates carrying a functional TAR RNA element and is strongly inhibited by low concentrations of 5,6-dichloro-1-β- d -ribofuranosyl benzimidazole (DRB), a potent inhibitor of CDK9, the protein kinase subunit of the Tat-associated kinase (TAK). Immunoblotting experiments have shown that CDK9 and its associated cyclin, cyclin T1, are present at equivalent levels in both the pre-initiation and elongation complexes. We conclude that activation of the CDK9 kinase, leading to CTD phosphorylation, occurs only in elongation complexes that have transcribed through the Tat-recognition element, TAR RNA.

We use a synthetic “primed bubble-duplex” model elongation complex developed previously to examine certain structural and thermodynamic features of the transcription elongation complex of Escherichia coli. The nucleic acid framework of this model complex consists of a linear base-paired DNA molecule with a central “bubble” of non-complementary nucleotide residues, together with a single-stranded RNA molecule that is complementary (at its 3′-end) to three to 12 nucleotide residues of one of the DNA strands within the bubble. RNA polymerase is added to this framework in trans, and on addition of rNTPs the resulting complex can elongate the 3′-end of the RNA primer in a template-dependent manner with functional properties that are indistinguishable from those of a “natural” promoter-initiated transcription elongation complex operating under the same conditions. In this study we use this model system to show that the formation of a stable elongation complex at any particular template position can be treated as an equilibrium process, and that semi-quantitative dissociation constants can be estimated for the complex by using a gel band-shift assay to monitor the binding of the RNA oligomer to the complex. We then show that the formation of a stable complex depends on the presence of a complementary RNA-DNA hybrid that is at least 9 bp in length, and in addition that several nucleotide residues of non-complementary RNA located upstream of the RNA-DNA hybrid bind strongly to the putative single-stranded RNA binding site of the polymerase and significantly enhance the stability of the resulting elongation complex. Finally, we demonstrate that the measured stabilities of the model constructs in which the length of the RNA-DNA hybrid is varied correlate well with the transcriptional processivity of the functioning complex that results when rNTPs are added. These findings are discussed in the context of related studies of both model systems and natural elongation complexes. The general concepts that emerge are used to define some central structural and functional features of the transcription complex.

The 64 amino acid residue product of bacteriophage T7 gene 2 (gp2) binds the Escherichia coli RNA polymerase and inhibits transcription. We localized the gp2 binding site to within 53 amino acid residues in the functionally dispensable region of the RNA polymerase beta' subunit. We investigated the effect of gp2 on transcription at a -10/-35 promoter and at an "extended -10" promoter. Our results indicate that binding of gp2 to the sigma70holoenzyme (Esigma70) prevents promoter recognition at -10/-35 promoters. Once open promoter complexes are formed, however, Esigma70transcription is resistant to gp2, since gp2 can no longer bind RNA polymerase. Surprisingly, transcription inhibition by gp2 is both sigma and promoter-specific. gp2 has little effect on Esigma70transcription from an extended -10 promoter, which does not depend on sigma70region 4 interactions with the -35 promoter box for its activity. gp55-dependent phage T4 late promoter transcription is also resistant to gp2. From these results, we conclude that the interaction of the sigma70region 4 with the -35 consensus promoter element is the primary target of gp2 inhibition.

The yeast TFIIIB transcription factor is composed of three components, TBP, TFIIIB90 or B″, and TFIIIB70 or BRF. TFIIIB70 is a pivotal component since it interacts with TBP, TFIIIC and RNA polymerase III (pol III). In order to better understand the role of TFIIIB70, we mutagenized extensively three evolutionary conserved motifs of its pol III-specific C-terminal extension. Conditional mutations lying in conserved regions II and III were obtained, some of which altered the interaction with the C34 subunit of pol III and were co-lethal with rpc34 mutations. Two conditional mutations in region II impaired the interaction with TBP and were suppressed by its overexpression. The pattern of suppression of the strongest mutation by overexpression of various mutant TBP, suggested a contact between TBP-R220 and TFIIIB70-D464 residues in vivo. As expected, this TFIIIB70 mutation impaired the assembly of TFIIIB·TFIIIC·DNA complexes and affected in vitro transcription of the SUP4 tRNA gene. Our results underscore the important role of region II of TFIIIB70 in pre-initiation as well as transcription complex assembly via C34 and TBP binding.

The "extended -10" motif described originally in Escherichia coli promoters occurs frequently in other bacterial promoters. Most Bacillus subtilis bacteriophage o29 promoters contain this motif. To analyse the influence of the motif on sigmaA-RNA polymerase transcription, the 5'-TG-3' dinucleotide was changed to 5'-AC-3' in three o29 promoters. This change impaired RNA polymerase binding to the promoters; the yields of closed and open complexes were reduced independently of other differences inherent to each promoter. The mutation abolished transcription in vitro from a promoter lacking the consensus sequence at the -35 hexamer. In contrast, at other promoters with a -35 consensus sequence, the yield of run off transcription was not reduced by the mutation. Indeed an apparent interference phenomenon at high polymerase/DNA ratios was relieved. These results indicate that the extended -10 motif provides contact points for sigmaA-RNA polymerase with a role restricted to the first steps of transcription.

The sigma subunit of RNA polymerase orchestrates basal transcription by first binding to core RNA polymerase and then recognizing promoters. Using a series of 16 alanine-substitution mutations, we show that residues in a narrow region of Escherichia coli sigma70 (590 to 603) are involved in transcription activation by a mutationally altered CRP derivative, FNR and AraC. Homology modeling of region 4 of sigma70 to the closely related NarL or 434 Cro proteins, suggests that the five basic residues implicated in activation are either in the C terminus of a long recognition helix that includes residues recognizing the -35 hexamer region of the promoter, or in the subsequent loop, and are ideally positioned to permit interaction with activators. The only substitution that has a significant effect on activator-independent transcription is at R603, indicating that this residue of sigma70 may play a distinct role in transcription initiation.

Activation of transcription at bacteriophage T4 late promoters and coupling of late transcription to concurrent replication requires a peculiar transcriptional activator, the gp45 sliding clamp of the T4 DNA polymerase. In order to activate transcription, the topologically DNA-linked trimeric gp45 must interact with two T4-encoded RNA polymerase-binding proteins, the gp33 co-activator, and the gp55 late sigma factor. The carboxy termini of gp55 and gp33 share a similar sequence, which has been shown to be required for response of late transcription to activation by gp45. Alanine-scanning mutagenesis of the C terminus of gp55 shows that residues within the short hydrophobic sequence L(D/A)FLYE, are necessary for gp55 to bind to gp45, and to respond maximally to transcriptional activation by gp45. When fused to GST, the peptide SLDFLYE suffices for specific gp45 binding. Thus, it constitutes the main gp55 epitope for gp45 interaction.

Entry into mitosis is accompanied by a global repression of transcription. To investigate the molecular mechanisms which shut-down rRNA synthesis during mitosis, we have compared RNA polymerase I (Pol I) transcription in extracts from asynchronous and mitotic HeLa cells. We show by several experimental approaches that phosphorylation by cdc2/cyclin B inactivates the TBP-containing factor SL1 and thus abrogates Pol I transcription during mitosis. This finding links the cell's cycle with the transcriptional activity of Pol I and suggests a common mechanism for mitotic silencing of all three classes of nuclear RNA polymerases, i.e. reversible inactivation of the respective TBP-TAF complexes by (a) mitotic kinase(s).

The TATA binding protein (TBP), which plays a central role in gene regulation as an essential component of all three nuclear transcription systems, sharply kinks the TATA box at two sites and severely contorts the intervening DNA segment. DNA constructs with precisely localized flexure have been used to investigate the special repertoire of mechanisms and properties that arise from TBP interacting with the TATA box. DNA flexure precisely localized to the sites of TBP-mediated DNA kinking increases the affinity of TBP more than 100-fold; unexpectedly, this increase in affinity is achieved almost exclusively by increasing the stability of the TBP-DNA complex rather than the rate of its formation. In vitro transcription with RNA polymerase III provides a first demonstration that the orientation of TBP on the TATA box is governed by DNA deformability, its C-proximal repeat contacting the more flexible end of the TATA box. Exceptionally stable TBP-DNA complexes reach their orientational equilibrium very slowly; in these circumstances, assembly of stable (“committed”) transcription initiation complexes can freeze far-from-equilibrium orientations of TBP on the TATA box, causing transcription polarity to be determined by a kinetic trapping mechanism.

The molecular mechanisms underlying transcription elongation and their role in gene regulation are poorly characterized in eukaryotes. A number of genes, however, have been proposed to be regulated at the level of transcription elongation, including c-myc, c-fos and c-myb. Here, we analyze the control of transcription elongation at the mouse c-fos gene at the nucleotide level in intact cells. We find that RNA polymerases are engaged in the promoter-proximal part of the gene in the absence of gene activation signals and mRNA synthesis. Importantly, we determine that the engaged RNA polymerases originate from a continuous initiation of transcription which, in the absence of gene activation signals, terminate close to the promoter. We also observe that the c-fos gene presents an active chromatin conformation, with the promoter and upstream regulatory sequences constitutively occupied by proteins, accounting for the continuous initiation of RNA polymerase complexes. We propose that activation of c-fos gene expression results primarily from the assembly of elongation-competent RNA polymerases that can transcribe the complete gene. Our results suggest that the engaged RNA polymerases found downstream of a number of other eukaryotic promoters may be associated with transcription termination of elongation-incompetent polymerases in the absence of activating signals.

We have examined the role of the TATA box in determining transcription initiation frequency in vitro by studying a collection of promoters containing different TATA sequences in the context of the adenovirus major late promoter. In addition to measuring transcription rates, we have determined how the sequence changes affected the association and dissociation kinetics and the affinity of TBP binding. We observed that transcription from promoters containing the highest affinity TATA boxes is limited by the rate with which TBP associates with the promoter. In contrast, transcription from promoters containing lower affinity TATA boxes appears to be limited both by how much TBP is bound and by the relatively low occupancy of the conformation that can undergo subsequent steps in preinitiation complex assembly. The implications of these results in understanding the mechanism of transcription enhancement by transcriptional activators is discussed.

We study here the effect on phage T7 RNA polymerase activity of lac repressor bound downstream of the T7 promoter. When repressor binds in vitro at an operator centered at +13 or +15 with respect to transcription start, it does not prevent initiation, though the transcript yield is reduced. However, the processivity of the polymerase is depressed and transcript extension is blocked at positions +4 and +6, respectively. These results indicate that repressor and polymerase do not simply exclude each other from the promoter. Rather, they would come into steric conflict and compete for establishment or retention of interactions with the same segment of DNA, without this leading to the immediate displacement of either polymerase or repressor. The resulting destabilization of the transcription complex would depress both initiation rate and enzyme processivity. In contrast to the above results, little reduction in runoff transcription is observed when operator is centered at +47. The decreased sensitivity of polymerase to repressor bound at +47 versus +13 or +15 is likely to be due to the higher stability of the elongation complex during the transcription of downstream regions in comparison with the first transcribed nucleotides. We also show that under conditions of leaky repression and with operator centered at +13, a mutant T7 RNA polymerase showing normal promoter affinity but a slower elongation rate is more sensitive to repression than the wild-type enzyme, both in vitro and in vivo. In vitro, this higher sensitivity is largely due to a reduced ability of the mutant to overcome the elongation block at position +4. The parallel between the in vitro and in vivo data suggests that in vivo the repressor also does not prevent polymerase from binding to promoter, but interferes with subsequent steps in initiation and transcript extension, in this case presumably largely extension beyond +4.

Regulatory protein p4 of Bacillus subtilis phage Φ29 activates transcription from the viral late A3 promoter by interacting with the C-terminal domain (CTD) of the B. subtilis RNA polymerase α subunit, thereby stabilizing the holoenzyme at the promoter. Protein p4 does not interact with the Escherichia coli RNA polymerase and cannot activate transcription with this enzyme. We have constructed a chimerical α subunit containing the N-terminal domain of the E. coli α subunit and the CTD of the B. subtilis α subunit. Reconstitution of RNA polymerases containing this chimerical α subunit, the E. coli β and β′ subunits, and the vegetative σ factor from either E. coli (σ70) or B. subtilis (σA), generated hybrid enzymes that were responsive to protein p4 and efficiently supported activation at the A3 promoter. Protein p4 activated transcription with the chimerical enzymes through the same activation surface used with B. subtilis RNA polymerase. Therefore, the B. subtilis α-CTD allowed activation by p4 even when the rest of the RNA polymerase subunits belonged to E. coli, a distantly related bacterium. These results strongly suggest that protein p4 works essentially by serving as an anchor that stabilizes RNA polymerase at the promoter.

By binding to the DNA site OE at position -60.5 in the gal operon, the GalR protein activates transcription from the P2 promoter located on the opposite face of DNA (position -5) and represses transcription from the P1 promoter located on the same face (position +1). GalR increases RNA polymerase binding at P2 and inhibits isomerization at P1 by forming a GalR-DNA-RNA polymerase ternary complex in each case. The specific effect of GalR at one promoter is independent of the presence of the other promoter. The enhancement or repression is also not the intrinsic property of a promoter; the regulation can be reversed by switching the angular orientation of the promoters relative to OE. Both enhancement and repression appear to require the same interaction between RNA polymerase alpha-subunit and GalR and/or the same interaction between RNA polymerase alpha-subunit and DNA in the ternary complexes. We have discussed how GalR might exert opposite effects in the steps involved in the formation of the open complex from free RNA polymerase and DNA.

Bacteriophage T7 lysozyme is known to inhibit transcription by T7 RNA polymerase. Lysozyme present before initiation inhibited the synthesis of long RNA chains but did not inhibit elongation when added shortly after chains were initiated. A combination of gel-shift and transcription assays showed that lysozyme and polymerase form a 1:1 complex that binds promoter DNA and makes abortive transcripts, indicating that lysozyme has little effect on the early steps of transcription. Extension of stalled transcription complexes suggested that a transcribing polymerase becomes resistant to lysozyme inhibition after synthesis of an RNA chain as short as 15 nucleotides. It seems likely that bound lysozyme prevents an initiating polymerase from converting to an elongation complex. This conversion is thought to involve both a conformational change in the polymerase and the binding of nascent RNA. Gel-shift experiments indicated that lysozyme does not interfere with the binding of RNA, so it probably prevents a necessary conformational change in the polymerase. Lysozyme also increased pausing or termination at two sites in lambda DNA and at a site near the right end of the concatemer junction of T7 DNA. If pausing at these sites involves a reversal from the elongation to the initiation conformation, lysozyme may increase pausing or termination by "locking in" the initiation conformation. The arrest of transcription complexes near promoters and near the right end of the concatemer junction almost certainly must relate to lysozyme's ability to stimulate replication, maturation and packaging of T7 DNA during T7 infection.

In a previous study, we explored the mechanisms of SNR6 gene activation by grafting a heterologous DNA-binding domain, GAL4-(1-147), to various components of the yeast RNA polymerase III transcription system. Here, we demonstrate that a modified SNR6 gene harboring GAL4-binding sites (UAS(G)-SNR6) can be efficiently activated via an intervening, unrelated protein-protein interaction, thus laying the foundations of a RNA polymerase III-based two-hybrid system. In a model system, the interacting proteins recruiting TFIIIC to DNA were PRP21 and PRP9 or PRP21 and PRP11. Mutations affecting the interaction between PRP21 and PRP9, or PRP21 and PRP11 decreased UAS(G)-SNR6 activation level proportionally. RNA polymerase II transcriptional activators, like GAL4, VP16 or p53, fused to GAL4 DNA-binding domain, did not activate the UAS(G)-SNR6 gene. However, GAL4 strongly activated UAS(G)-SNR6 when GAL80, an interacting protein, was fused to TFIIIC. This result indicates that this two-hybrid system can be used to assess the interactions between RNA polymerase II regulatory proteins and their partners.

Sporulation in Bacillus subtilis is dependent on the response regulator Spo0A, which both represses and activates transcription in vitro. The activity of Spo0A is increased by phosphorylation. We previously demonstrated that the phosphorylation increased the ability of Spo0A to stimulate in vivo transcription from the promoter for the spoIIG operon, one of the operons known to be regulated by Spo0A in vivo. In the work reported here we have examined the kinetics of transcription initiation at the spoIIG operon promoter using a single round transcription assay and the kinetics of formation of spoIIG promoter-RNA polymerase complexes using DNase I footprinting. Both the kinetic assays and the footprint assays indicated that the initial binding of the polymerase to the template was not dependent on the presence of Spo0A. The phosphorylated form of Spo0A stimulated the rate of initiation by affecting a step that occurred after the initial interaction of the polymerase with the template. Phosphorylation of Spo0A may stimulate transcription by modifying preinitiation complexes containing the polymerase and the promoter.

Mutants of bacteriophage T7 RNA polymerase defective in functions other than transcription were sought by random chemical mutagenesis of the cloned gene and selection for inability to support the growth of a T7 mutant whose growth is dependent on T7 RNA polymerase supplied by the host cell. About half of the mutant clones appeared unable to make full-length T7 RNA polymerase, many of them producing a truncated protein. Among 116 mutants expressing full-length protein, two-thirds were severely impaired in transcription, but a surprisingly high one-third were able to direct significant transcription in vivo. Both types of mutation were distributed across much of the gene, as determined by a rapid genetic mapping procedure that allows the lethal mutation in each clone to be localized. One mutation (isolated twice) allowed normal gene expression but prevented the formation of mature ends of T7 DNA from concatemers, which normally happens during packaging into phage particles. Thirty-seven of the mutations appeared to increase the sensitivity of the polymerase to inhibition by T7 lysozyme; all were suppressed by mutations in the lysozyme gene, including one suppressor constructed to retain full amidase activity but to be unable to bind T7 RNA polymerase. The two lysozyme-hypersensitive polymerase mutants analyzed in detail showed premature cessation of transcription during infection. Early proteins and those late proteins specified by genes as far right in T7 DNA as genes 8-9 appeared to be produced normally, but expression of genes farther to the right was strongly depressed. DNA replication was depressed about 50% in one of these mutants and 90% in the other, even though the T7 replication proteins were made in normal amounts at the normal time.

Kin28p, associated with cyclin Ccl1p, is a putative cyclin-dependent kinase (CDK) of the p34cdc2 family in Saccharomyces cerevisiae. Search for mutations co-lethal (syn mutations) with a kin28 thermosensitive mutation (kin28-ts3) has uncovered genetic interactions between gene KIN28 and genes RAD3, SIN4, STI1 and CDC37. The genetic interaction between KIN28 and the CDC37 cell division cycle gene suggests that a connection exists between the activity of CDK-Kin28p and cell-cycle progression. Both RAD3 and SIN4 gene products are implicated in the RNA polymerase II transcription process. Here we show that RNA polymerase II transcription is drastically reduced in a kin28-ts mutant, at restrictive temperature. This impairment correlates with a markedly decreased phosphorylation of the C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Rpb1p). Thus, the Kin28 gene product is required in vivo for RNA polymerase II phosphorylation and transcriptional activity as recently suggested by experiments using an in vitro reconstituted system.

Genetic and molecular analysis in Drosophila melanogaster identifies eight suppressor mutations in the second largest subunit of RNA polymerase II. The suppressor mutations fall into two classes: five are strong, result from the same serine to cysteine amino acid residue substitution and rescue one conditional lethal allele in the largest subunit of RNA polymerase II; there are mild, result from a change in the same methionine residue to either isoleucine or valine, are located seven amino acid residues away from the strong suppressors and rescue two conditional lethal alleles in the largest subunit. Sequence analysis of the three regions around these mutations demonstrates that they are located within highly conserved domains but fails to explain the observed genetic interactions. One of the conditional lethal alleles maps within a region previously reported to share sequence similarity to Escherichia coli DNA polymerase I. As the gross structure of RNA polymerase II and DNA polymerase I is similar, even though their primary sequence is not, we predict that more similarities exist but may be too highly divergent to be detected by normal homology searches. We indentify the most similar regions between each of the three conserved domains of RNA polymerase II, indentified as functionally important because of the mutations we isolated, and DNA polymerase I. Molecular modeling theses regions of RNA polymerase II onto the tertiary structure of DNA polymerase I predicts that all lie adjacent to the DNA binding cleft in positions such that they could interact with the phosphate backbone od DNA. This juxtaposition of mutations in two largest subunits of RNA polymerase II suggest a mechanism for their genetic interactions.

RNA polymerase II encounters various obstacles to transcript elongation both in vivo and in vitro. These include DNA sequence elements and protein bound to the major groove of DNA. Elongation factor SII binds to RNA polymerase II and enables the enzyme to bypass these impediments. SII also activates nascent RNA cleavage by the arrested transcription elongation complex, an activity intimately involved in the readthrough process. Here we identify another type of reversible blockage to RNA polymerase II transcription, the antitumor antibiotic distamycin, which binds in the minor groove of A + T-rich DNA. SII facilitates readthrough of arrest sites resulting from DNA-binding of the drug. In response to SII, these complexes cleave their nascent RNA chains. These findings confirm that SII is a general elongation factor that potentiates transcription through a variety of impediments. They also strengthen the idea that SII stimulates transcription by activating nascent RNA cleavage. In some cases, distamycin can potentiate transcription through a naturally occurring pause site. We also show that the template undergoes a conformational change in the presence of distamycin. This suggests that distamycin can transform DNA from an elongation-non-permissive configuration into an elongation-permissive form and we take this as independent evidence confirming that DNA structure influences transcription elongation by RNA polymerase II.

The analysis of RNA chain elongation by Saccharomyces cerevisiae RNA polymerase (Pol) III in the accompanying paper has been extended by examining the encounter of highly purified RNA polymerase with purified individual transcription factors. Arrested ternary transcription complexes were formed with purified Pol III initiating precisely at the 3' overhanging ends of linear DNA. Transcription factors were then bound to DNA and their effects on individual steps of RNA chain elongation were analyzed. The outcome of the encounter between Pol III and TFIIIC was orientation-specific. For RNA synthesis in the sense direction, with Pol III approaching the obstructing protein from the direction of normal transcription, pure TFIIIC rapidly yielded the way to the advancing polymerase: only a single step of RNA chain elongation was slightly slowed by pure TFIIIC occupying its boxB binding site in the SUP4 tRNA(Tyr) gene. In a complete cell-free fraction, protein binding to this tRNA gene likewise generated a delay of only approximately 0.15 to 0.2 second in executing the same step. Transcription by pure Pol III in the sense direction also dissociated the TFIIIC-SUP4 gene complex. The encounter of Pol III elongating RNA chains in the anti-sense direction with the backside of TFIIIC yielded a different outcome. RNA chain elongation paused extensively six to nine base-pairs beyond the downstream edge of the DNA-binding site of TFIIIC, with a median delay of nine seconds, approximately 50 times longer than in the sense direction. At the height of its effect on RNA chain elongation, the TFIIIC-imposed barrier entrapped the great majority of RNA chains, but their elongation was eventually allowed to continue. In contrast, DNA-bound TFIIIB completely blocked RNA chain elongation in the anti-sense direction. The role of the internal promoter element in transcription by Pol III is discussed in the light of this analysis. The large bulk of TFIIIC, which binds with high affinity to boxB, and also to boxA, is particularly suited to occluding its transcription unit to other proteins. At the same time, TFIIIC makes way for transcription so rapidly that it places no limit on the level of gene activity.

Dimethyl sulfate, DNase I and micrococcal nuclease DNA cleavage were combined with the ligation-mediated polymerase chain reaction to obtain high resolution maps of the promoter regions for two cell-type-specific genes: the a-specific STE2 gene and the alpha-specific STE3 gene. We find that MCM1 binds in vivo in a-cells to a 16 bp P-box sequence located in the STE2 UAS. In alpha-cells, the footprint pattern is extended relative to a-cells, consistent with the additional binding of MAT alpha 2 to the sequences flanking each end of the P-box. A nucleosome was found adjacent to the P-box of the transcriptionally repressed a-specific STE2 UAS in alpha-cells, positioned so that the nucleosome overlaps the TATA-box. In contrast, such well-positioned nucleosomes were not found for the transcriptionally active STE2 UAS in a-cells, where instead the TATA box appears to be bound to the general transcription factor TFIID. These observations support the hypothesis that MAT alpha 2 repression of a-specific genes is mediated by nucleosomes, perhaps by exclusion of TFIID from the TATA-box.