The general transcription factor TFIIE plays essential roles in both transcription initiation and the transition from initiation to elongation. Previously, we systematically deleted the structural motifs and characteristic sequences of the small subunit of human TFIIE (hTFIIE␤) to map its functional regions. Here we introduced point mutations into two regions located near the carboxy terminus of hTFIIE␤ and identified the functionally essential amino acid residues that bind to RNA polymerase II (Pol II), the general transcription factors, and single-stranded DNA. Although most residues identified were essential for transcription initiation, use of an in vitro transcription assay with a linearized template revealed that several residues in the carboxyterminal helix-loop region are crucially involved in the transition stage. Mutations in these residues also affected the ability of hTFIIE␤ to stimulate TFIIH-mediated phosphorylation of the carboxy-terminal heptapeptide repeats of the largest subunit of Pol II. Furthermore, these mutations conspicuously augmented the binding of hTFIIE␤ to the p44 subunit of TFIIH. The antibody study indicated that they thus altered the conformation of one side of TFIIH, consisting of p44, XPD, and Cdk-activating kinase subunits, that is essential for the transition stage. This is an important clue for elucidating the molecular mechanisms involved in the transition stage.In eukaryotes, the expression of protein-coding genes is strictly regulated at the level of transcription by RNA polymerase II (Pol II). Once signals from outside the nucleus are received and the condensed form of the inactive chromatin is activated and remodeled by chromatin-modulating factors, five general transcription factors (TFIIB, TFIID, TFIIE, TFIIF, and TFIIH) together with Pol II form the preinitiation complex (PIC) on the core promoter. Formation of this complex is assisted by various transcriptional activators, cofactors, and mediators (for reviews, see references 19, 25, and 43). Analyses of the PIC assembly pathway using isolated general transcription factors have revealed that the factors can assemble stepwise in vitro. This process commences with the binding of TFIID to the TATA box on the core promoter and ends with TFIIE and TFIIH joining the PIC (reviewed in references 10, 26, 34, and 42). It is widely accepted that TFIIE and TFIIH stabilize and activate the PIC by binding to all the other general transcription factors as well as to Pol II and at the same time open up the double-stranded DNA (dsDNA) at the region from Ϫ9 to ϩ2, adjacent to the transcription initiation site (ϩ1), in a manner that is dependent on dATP hydrolysis (14,56). This process is known as promoter melting. These various functions of TFIIE and TFIIH have been revealed recently by three types of studies. First, photo-cross-linking studies demonstrated that TFIIE␤ binds directly to the core promoter region between positions Ϫ14 and Ϫ2, which is where the promoter melts upon transcription initiation (5, 41). Second, two-dimensiona...

The human t hyroid hormone r eceptor-a ssociated p roteins (TRAP)/Mediator and related complexes mediate transcription through regulatory factors. To further understand the structural and functional diversity of these complexes we established three HeLa cell lines each expressing one of three epitope-tagged human TRAP/Mediator subunits, MED6, MED7, and CDK8 and isolated the complexes in which these subunits were contained by affinity and HPLC-gel filtration chromatography. The largest complexes from each cell line had a molecular mass of 1.5 MDa and possessed almost identical subunit compositions; we designated these complexes T RAP/ M ediatorl ike c omplex 1 (TMLC1). Two potential subcomplexes were additionally observed: a 1-MDa complex from the CDK8-cell line (TMLC2) and a 600-kDa complex from the MED6-cell line (TMLC3). All three complexes regulated transcription in vitro ; TMLC1 and TMLC3 augmented transcriptional activation, whereas TMLC2 repressed it. TMLC1 and TMLC2 phosphorylated RNA polymerase II (Pol II), but TMLC3 did not. Furthermore, TMLC1 predominantly interacted with the general transcription factors TFIIE, TFIIF, and TFIIH, which function during transcription initiation and the transition to elongation. In a final experiment, knockdown of CDK8 using RNA interference prevented transcriptional activation by Gal4-VP16 in a luciferase-assay. This, together with the effect of TMLC1 on transcription in vitro , suggests that CDK8 play positive roles in transcriptional activation.

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