We report here the isolation of a human RNA polymerase II complex containing a subset of the basal transcription factors and the human homologues of the yeast SRB (for suppressors of RNA polymerase B) proteins. The complex contains transcriptional coactivators and increases the activation of transcription. In addition, some components of the RNA polymerase II complex participate in DNA repair.

A central issue in eukaryotic transcriptional regulation is the mechanism by which promoter-specific transcription factors (activators) stimulate transcription. Two lines of evidence indicate that the general transcription factor TFIIB is a pivotal component in the mechanism by which an acidic activator functions. First, during assembly of the preinitiation complex TFIIB binding is a rate-limiting step enhanced by an acidic activator. Second, the TFIIB activity in a HeLa cell nuclear extract is specifically retained on a column containing an acidic activating region. But because our previous study monitored only TFIIB activity, it remains possible that the interaction between TFIIB and the acidic activating region is mediated through additional proteins, for example, those designated as adaptors, coactivators or mediators. A complementary clone encoding TFIIB has recently been isolated and shown to encode a polypeptide of relative molecular mass 35,000. Here we report that TFIIB expressed in and purified from Escherichia coli (recombinant TFIIB) binds directly to the potent acidic activating region of the herpes simplex virus-1 VP16 protein.

ABSTRACT5-Capping is an early mRNA modification that has important consequences for downstream events in gene expression. We have isolated mammalian cDNAs encoding capping enzyme. They contain the sequence motifs characteristic of the nucleotidyl transferase superfamily. The predicted mouse and human enzymes consist of 597 amino acids and are 95% identical. Mouse cDNA directed synthesis of a guanylylated 68-kDa polypeptide that also contained RNA 5-triphosphatase activity and catalyzed formation of RNA 5-terminal GpppG. A haploid strain of Saccharomyces cerevisiae lacking mRNA guanylyltransferase was complemented for growth by the mouse cDNA. Conversion of Lys-294 in the KXDG-conserved motif eliminated both guanylylation and complementation, identifying it as the active site. The K294A mutant retained RNA 5-triphosphatase activity, which was eliminated by N-terminal truncation. Full-length capping enzyme and an active C-terminal fragment bound to the elongating form and not to the initiating form of polymerase. The results document functional conservation of eukaryotic mRNA guanylyltransferases from yeast to mammals and indicate that the phosphorylated C-terminal domain of RNA polymerase II couples capping to transcription elongation. These results also explain the selective capping of RNA polymerase II transcripts.Addition of a 5Ј-terminal cap is an important, early event in mRNA formation (1). This structural hallmark of most eukaryotic mRNAs enhances splicing (2-4), transport (5), translation (6), and stability (7,8) and is essential for viability (9).Caps are formed on nascent nuclear pre-mRNAs by conversion of 5Ј-tri-diphosphate to 5Ј-diphosphate ends, followed by addition of GMP and methylation (1, 10). The guanylyltransfer reaction characterized in various systems involves formation of an active enzyme intermediate containing GMP covalently attached to lysine (11). In yeast, mRNA capping enzyme consists of separate subunits for RNA 5Ј-triphosphatase and guanylyltransferase activities (9, 12). cDNA clones coding for mRNA guanylyltransferase in Saccharomyces cerevisiae (9), Schizosaccharomyces pombe (13), and Candida albicans (14) have been sequenced. Each contains the active site lysine in KXDG (13, 15), one of several highly conserved motifs characteristic of a superfamily of nucleotidyl transferases (16). A number of viral capping enzymes also contain these diagnostic sequence motifs, and the recently solved structure of capping enzyme from Chlorella virus PBCV-1 suggests that specific residues in these motifs are important for binding GTP (17). Despite this detail of sequence and structure information, no metazoan capping enzyme previously has been cloned and characterized.To explore the molecular interactions that result in selective capping of RNA polymerase II (pol II) transcripts in mammalian cells, we have isolated and characterized cDNA clones that code for the human and mouse capping enzymes. Functional studies demonstrated that the mammalian enzyme complements the lethality of a S. cerevisiae mu...

Human transcription factor TFIID, the TATA-binding protein, was partially purified to a form capable of associating stably with the TATA motif of the adenovirus major late promoter. Binding of the human and yeast TFIID to the TATA motif was stimulated by TFIIA. TFIIA is an integral part of a complex capable of binding other transcription factors. A complex formed with human TFIID and TFIIA (DA complex) was specifically recognized by TFIIB. We found that TFIIB activity was contained in a single polypeptide of 32 kDa and that this polypeptide participated in transcription and was capable of binding to the DA complex to form the DAB complex. Formation of the DAB complex required TFIIA, TFIID, and sequences downstream of the transcriptional start site; however, the DA complex could be formed on an oligonucleotide containing only the adenovirus major late promoter TATA motif. Using anti-TFIIB antibodies and reagents that affect the stability of a transcription-competent complex, we found that yeast and human TFIID yielded DAB complexes with different stabilities.The promoters of genes transcribed by RNA polymerase II are composed of different cis-acting sequence elements that participate in the regulation of expression (23). Two of these elements, the TATA motif, which is located approximately 30 nucleotides upstream of the transcriptional start site (1), and the initiator (34), which .encompasses the start site of transcription, are present in many genes. These two transcriptional control elements appear to be recognized by a complex set of transcription factors (for reviews, see references 24 and 30). The factors operating through these elements are required for transcription of all genes thus far studied, including those that do not contain a TATA motif, and therefore they have been classified as general or basal transcription factors. Of the five general transcription factors (TFIIA, -IIB, -IID, -IIE, and -IIF) that have been described, TFIID appears to be the only factor with an associated DNA-binding activity with specificity for the TATA motif (6,9,18,20,25,28,35). The other factors and RNA polymerase II appear to associate with promoter sequences primarily by protein-protein interactions. Indeed, it has been demonstrated that TFIIE and TFIIF can independently form a stable complex (the TFIIE/F complex) with RNA polymerase II (10, 11). Moreover, it has been suggested that a protein fraction containing TFIIE/F can form a complex with TFIIB (29).The human TFIID (hTFIID) has been difficult to purify. A breakthrough in the field was the discovery that yeast cells contained a TFIID activity (yTFIID) that could functionally substitute for the hTFIID in transcription systems reconstituted with all human factors (3,5). The yTFIID activity has been purified to homogeneity; these studies demonstrated that the yTFIID activity is contained in a single polypeptide of 27 kDa (20). The reported molecular weight of the yeast factor was confirmed with the isolation of the yTFIID gene (4,8,14,19,33). Isolation of the yTFIID gen...

How eukaryotic promoter-specific activator proteins (activators) stimulate transcription is a central question. We have previously shown that an acidic activator can directly interact with the general transcription factor TFIIB and increase its stable assembly into a preinitiation complex. We have proposed that this increase in TFIIB assembly is at least part of the mechanism by which an acidic activator functions. A prediction of this hypothesis is that a TFIIB mutant unable to interact with an acidic activator could not support activated transcription, and here we present experiments that verify this prediction. In conjunction with previous studies, our results argue that interaction between an acidic activator and TFIIB is necessary for transcriptional activation.

Transcription factor IIB (TFIIB) plays a pivotal role in the formation of transcription-competent initiation complexes. TFIIB was found to interact with the TATA-binding protein, the small subunit of TFIIF, and RNA polymerase II. These interactions require distinct domains in THIB. Using the gel mobility-shiit assay, it was found that the amino terminus of TFIIB was necessary for the formation of complexes containing RNA polymerase II and TFIIF, whereas the carboxy-terminal domain, which is composed of two imperfect direct repeats and includes a putative amphipathic a-helix, was sufficient for the formation of complexes containing the TATA-binding protein and TFIIB (DB complex). Protein-protein interaction analyses demonstrate that the amphipathic a-helix in TFIIB is important for the interaction with the TATA-binding protein. Specific residues mapping to the carboxyl terminus of the second direct repeat were found to be crucial for the interaction of TFIIB and RNA polymerase II. The interaction with the small subunit of TFIIF was mapped to the amino terminus of TFIIB, which includes a zinc finger. Identification of the protein factors that govern transcription initiation by RNA polymerase II has led to the delineation of two functional groups of transcription factors. One group, the general transcription factors (GTFs), catalyze basal transcription through the TATA and initiator DNA elements in a multiprotein complex with RNA polymerase II {RNAPII} (for review, see Weis and Reinberg 1992~ Zawel and Reinberg 1992}. The second group, the specific factors, is composed of DNA-binding proteins that bind independently of RNAPII to specific DNA sequences upstream of the TATA and initiator DNA elements (for review, see Johnson and McKnight 1989~ Mitchell andTjian 1989}. The specific transcription factors are thought to participate directly in regulatory networks that mediate either stimulation or repression of basal transcription of specific genes. It is likely that the specific transcription factors exert their modulatory actions on basal transcription by interacting directly with the GTFs and RNAPII {for review, see aCorresponding author.

Adenovirus ElA has long been known to activate/repress cellular and viral transcription. The transcriptional activity of nuclear extracts was depleted after chromatography on immobilized ElA protein columns that specifically retained the transcription factor (TF) IID. 5124The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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