Abstract
The general transcription factor TFIIE plays important roles at two distinct but sequential steps in transcription as follows: preinitiation complex formation and activation (open complex formation), and the transition from initiation to elongation. The large subunit of human TFIIE (TFIIEalpha) binds to and facilitates the enzymatic functions of TFIIH, but TFIIE also functions independently from TFIIH. To determine functional roles of the small subunit of human TFIIE (TFIIEbeta), deletion mutations were systematically introduced into putative structural motifs and characteristic sequences. Here we show that all of these structures that lie within the central 227-amino acid region of TFIIEbeta are necessary and sufficient for both basal and activated transcription. We further demonstrate that two C-terminal basic regions are essential for physical interaction with both TFIIEalpha and single-stranded DNA, as well as with other transcription factors including the Drosophila transcriptional regulator Krüppel. In addition, we analyzed the effects of the TFIIEbeta deletion mutations on TFIIH-dependent phosphorylation of the C-terminal domain of RNA polymerase II and on wild type TFIIEbeta-driven basal transcription. Both responsible regions also mapped within the essential 227-amino acid region. Our results suggest that TFIIE engages in communication with both transcription factors and promoter DNA via the TFIIEbeta subunit.
Highlights
In eukaryotes productive transcription initiation by RNA polymerase II (Pol II)1 plays a key role in the regulation of gene expression in response to various developmental and environ
Initiation by Pol II requires five general transcription factors (TFIIB, TFIID, TFIIE, TFIIF, and TFIIH) that act through core promoter elements and is regulated by tissueand/or gene-specific factors that act through distal control elements
Elucidation of the precise mechanisms involved in transcription initiation by Pol II has been a long-standing issue in molecular biology
Summary
In eukaryotes productive transcription initiation by RNA polymerase II (Pol II) plays a key role in the regulation of gene expression in response to various developmental and environ-. Analysis of the PIC assembly pathway using isolated factors has revealed that this process begins with the binding of TBP (the TATA-binding protein) component of TFIID to the TATA box This is followed by the sequential interactions of TFIIB, a complex containing Pol II and TFIIF, TFIIE, and TFIIH. Human TFIIH consists of 9 subunits, and surprisingly, some of these subunits have been implicated in nucleotide excision repair and cell cycle regulation (for a review, see Ref. 11) This multisubunit general transcription factor is quite unique in that it contains the following three ATP-dependent catalytic activities: a kinase activity that phosphorylates the CTD (C-terminal domain) of the largest subunit of Pol II, a DNA-dependent ATPase, and a DNA helicase. This paper is available on line at http://www.jbc.org cruiting TFIIH into the PIC and for the modulation of the two functions of this factor in transcription initiation and in the transition from initiation to elongation
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