Studies of transcription by RNA polymerase II have revealed two promoter elements, the TATA motif and the initiator (Inr), capable of directing specific transcription initiation. Although binding to the TATA motif by one of the components of the transcription machinery has been shown to be the initial recognition step in transcription complex formation, many promoters that lack a traditional TATA motif have recently been described. In such TATA-less promoters, the Inr element is critical in positioning RNA polymerase II. Various Inr elements have been described and classified according to sequence homology. These Inr elements are recognized specifically by Inr-binding proteins. Interaction between these Inr-binding proteins and components of the basal transcription machinery provides a means through which a transcription competent complex can be formed.

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...

Two promoter elements, the TATA element and initiator (Inr), are capable of directing specific transcription initiation of protein-encoding genes by RNA polymerase II (RNAPII). Although binding to the TATA element by the TATA-binding protein (TBP) has been shown to be the initial recognition step in transcription complex formation in vitro, the mechanism through which the basal machinery assembles into a functional complex on TATA-less promoters is controversial. Evidence supporting numerous models of Inr-mediated transcription complex formation exists, including the nucleation of a complex by Inr-binding proteins, a component of the TFIID complex, or a specific upstream activator common to many TATA-less promoters, Sp1. Using various techniques, we have undertaken a systematic analysis of the natural TATA-less human DNA polymerase ␤ (␤-pol) gene promoter. Although the ␤-pol promoter contains upstream Sp1 elements and a functional Inr that binds YY1, neither of these factors is essential for Inr-mediated transcription complex formation. A complex containing TBP, TFIIB, TFIIF, and RNAPII (DBPolF complex) is capable of forming on the promoter in an Inr-dependent manner. A single point mutation within the Inr that affects DBPolF complex formation diminishes ␤-pol transcriptional activity.Transcription initiation of protein-encoding genes is a complex process requiring the precise positioning of RNA polymerase II (RNAPII) on promoter DNA. This is accomplished via a series of interactions between RNAPII and at least six accessory proteins, TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH, termed the general transcription factors (GTFs) (37, 70). Additionally, nucleation of an initiation complex occurs by recognition of a specific core element (31,54,66). In many genes, the TATA element is the primary core element responsible for positioning the basal transcription machinery on the promoter. TFIID, a multisubunit protein complex consisting of the TATA-binding protein (TBP) and TBP-associated factors (TAFs), nucleates the formation of the transcription initiation complex by binding to the TATA element (8,22,59). In vitro experiments have shown that preinitiation complex assembly proceeds by sequential recruitment of TFIIB followed by RNAPII together with TFIIF, TFIIE, and TFIIH to the promoter-bound TFIID. This multistep model has recently been challenged by the isolation of an RNAPII complex containing a subset of the GTFs, SRB (suppressor of RNAP B) proteins, and additional polypeptides capable of interacting with each other in the absence of promoter DNA (29). Under the appropriate conditions, this complex is capable of specifically initiating transcription and mediating a response to transcriptional activators in vitro (30, 38).As more promoters of protein-encoding genes have been characterized, it has been found that many lack a TATA element (31,54,66). Studies using the TATA-less murine terminal deoxynucleotidyltransferase (TdT) promoter identified a second core element, the initiator (Inr), which encompasses the tra...

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A procedure to immunoaffinity purify the human transcription factor IIH (TFIIH) was developed using a monoclonal antibody that recognizes an epitope in ERCC3 (XPB), the largest subunit of TFIIH. The epitope recognized by the monoclonal antibody was mapped to 20 amino acids. A peptide containing the epitope was capable of displacing TFIIH from an immunoaffinity column containing the monoclonal antibody. The immunoaffinity purification procedure described allows a simple and efficient method to purify both the "core" and "holo" TFIIH complexes.Initiation of transcription by RNA polymerase II (RNAPII) is a complex process requiring the assistance of five factors known as the general transcription factors (GTFs). These are transcription factor (TF) 1 IIB, TFIID, TFIIE, TFIIF, and TFIIH (1, 2). TFIID is a multisubunit complex that includes the TATA binding protein (TBP) (3). TBP, in its recombinant form or as part of the TFIID complex, recognizes the TATA motif present in many genes and can nucleate the formation of transcriptioncompetent complexes (4). The TBP (TFIID)-TATA complex provides the foundation for the entry of TFIIB, which is then followed by the entry of RNA polymerase II. RNA polymerase II is escorted to the promoter by TFIIF (5). The resulting DNAprotein complex, TBPolF, contains RNA polymerase II stably associated with the promoter (2). This complex, however, is unique with respect to complexes formed with other RNA polymerases since the formation of an RNA polymerase II transcription-competent complex requires the function of two other GTFs, TFIIE and TFIIH (6).TFIIE is a tetrameric factor composed of two subunits, p56 and p34 (7,8). TFIIE interacts with RNA polymerase II (9) and TFIIH (10,11). It is currently unknown whether TFIIE enters the transcription complex with RNA polymerase or in a subsequent step with TFIIH. However, it is clear that the association of TFIIE with the TBPolF complex is necessary for the entry of TFIIH to the transcription complex (6). Recent studies have suggested an alternative model for the formation of transcription competent complexes. These studies suggest that the GTFs and RNA polymerase II are preassembled in an RNA polymerase holoenzyme complex (12)(13)(14).TFIIH also known as BTF2 was initially purified as a factor required to reconstitute accurate transcription by RNA polymerase II in vitro (6,15). BTF2 was originally presumed to be TFIIE (16). However, the availability of antibodies against a subunit of TFIIE (17) revealed that BTF2 and TFIIE are distinct (18). Furthermore, antibodies generated against the 62-kDa subunit of BTF2 revealed that BTF2 and TFIIH are the same factor (18,19). Additionally, an activity isolated from rat liver extracts, referred to as factor ␦, contained a subunit composition and properties similar to human TFIIH (20). Purification of TFIIH demonstrated that it is a multisubunit factor composed of nine polypeptides (6,15,20). The studies with the rat liver-derived factor were the first to demonstrate that factor ␦ (TFIIH) copurified wit...