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 general transcription factor TFIIE plays important roles in transcription initiation and in the transition to elongation. However, little is known about its function during these steps. Here we demonstrate for the first time that TFIIH-mediated phosphorylation of RNA polymerase II (Pol II) is essential for the transition to elongation. This phosphorylation occurs at serine position 5 (Ser-5) of the carboxy-terminal domain (CTD) heptapeptide sequence of the largest subunit of Pol II. In a human in vitro transcription system with a supercoiled template, this process was studied using a human TFIIE (hTFIIE) homolog from Caenorhabditis elegans (ceTFIIE␣ and ceTFIIE␤). ceTFIIE␤ could partially replace hTFIIE␤, whereas ceTFIIE␣ could not replace hTFIIE␣. We present the studies of TFIIE binding to general transcription factors and the effects of subunit substitution on CTD phosphorylation. As a result, ceTFIIE␣ did not bind tightly to hTFIIE␤, and ceTFIIE␤ showed a similar profile for binding to its human counterpart and supported an intermediate level of CTD phosphorylation. Using antibodies against phosphorylated serine at either Ser-2 or Ser-5 of the CTD, we found that ceTFIIE␤ induced Ser-5 phosphorylation very little but induced Ser-2 phosphorylation normally, in contrast to wild-type hTFIIE, which induced phosphorylation at both Ser-2 and Ser-5. In transcription transition assays using a linear template, ceTFIIE␤ was markedly defective in its ability to support the transition to elongation. These observations provide evidence of TFIIE involvement in the transition and suggest that Ser-5 phosphorylation is essential for Pol II to be in the processive elongation form.In eukaryotes, transcription of protein-encoding genes by RNA polymerase II (Pol II) is the first step in expression of those genes (for reviews, see references 4, 35, 44, and 51). Two sequential stages are now recognized in the establishment of Pol II processivity: transcription initiation and the transition from initiation to elongation. At initiation, five general transcription factors (TFIIB, TFIID, TFIIE, TFIIF, and TFIIH) together with Pol II form the preinitiation complex (PIC) on the core promoter. Two models of PIC formation have been proposed on the basis of recent analyses. One model involves stepwise association of the general transcription factors and Pol II on promoter DNA, while the other model entails promoter sequences binding to a preassembled Pol II holoenzyme that contains most of the general transcription factors as well as SRB (suppressor of RNA polymerase B)-and Med-containing complex (reviewed in references 3 and 22). In vitro analyses of stepwise assembly of the PIC using purified factors have demonstrated that TFIIE joins the complex at a position near the transcription start site (between positions Ϫ14 and Ϫ2), after Pol II and TFIIF have joined the complex (25, 49). TFIIE then recruits TFIIH, and these two factors stabilize and activate the PIC, resulting in isomerization of double-stranded (ds) promoter DNA (promoter me...

The ubiquitin-proteasome pathway is a major protein degradation pathway whose dysfunction is now widely accepted as a cause of neurodegenerative diseases, including Alzheimer's disease. Here we demonstrate that the F-box and leucine rich repeat protein2 (FBL2), a component of the E3 ubiquitin ligase complex, regulates amyloid precursor protein (APP) metabolism through APP ubiquitination. FBL2 overexpression decreased the amount of secreted amyloid ␤ (A␤) peptides and sAPP␤, whereas FBL2 mRNA knockdown by siRNA increased these levels. FBL2 overexpression also decreased the amount of intracellular A␤ in Neuro2a cells stably expressing APP with Swedish mutation. FBL2 bound with APP specifically at its C-terminal fragment (CTF), which promoted APP/CTF ubiquitination. FBL2 overexpression also accelerated APP proteasome-dependent degradation and decreased APP protein localization in lipid rafts by inhibiting endocytosis. These effects were not observed in an F-box-deleted FBL2 mutant that does not participate in the E3 ubiquitin ligase complex. Furthermore, a reduced insoluble A␤ and A␤ plaque burden was observed in the hippocampus of 7-month-old FBL2 transgenic mice crossed with double-transgenic mice harboring APPswe and PS1 M146V transgenes. These findings indicate that FBL2 is a novel and dual regulator of APP metabolism through FBL2-dependent ubiquitination of APP.

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