Abstract
Transcription factor IIB (TFIIB) recruits RNA polymerase II to promoters and inserts a finger domain into its active site, with unknown consequences. Here we show that that the tip of this finger is important for two transcription initiation functions. First, TFIIB acts as a catalytic cofactor for initial RNA bond formation. It does so via a pair of fingertip aspartates that can bind magnesium, placing TFIIB within a family of proteins that insert finger domains to alter the catalytic functions of RNA polymerase. Second, the TFIIB fingertip mediates the timing of the release of TFIIB that is associated with appropriate promoter escape. These initiation requirements may assist in RNA quality control by minimizing functional synthesis when RNA polymerase becomes inappropriately associated with the genome without having been recruited there by TFIIB.
Highlights
Transcription factor IIB (TFIIB) is unique among the general transcription factors in that it recycles during continuous transcription, i.e. TFIIF travels with the elongating RNA polymerase, and TATA-binding protein, TFIIH, and TFIIE largely remain bound to the promoter [5]
The data show that two aspartates in this region are required to power catalysis during initiation of RNA synthesis, i.e. the initiating RNA polymerase is not capable of efficient RNA bond formation in the absence of this assistance
Chemical probing data suggest that these aspartates can bind magnesium, which may confer the potential to reconfigure the active site of the enzyme
Summary
Mutagenesis of TFIIB—The glutathione S-transferase-tagged expression construct for human TFIIB was generously provided to us by Michael Carey (UCLA). The transcription reaction contained 200 ng of Gal4-AH (Protein One), 60 g of depleted HeLa nuclear extract, 5 ng of recombinant TFIIB, and 50 ng of linear DNA template in a total volume of 25 l. Each reaction of blocked beads was incubated with 200 ng of Gal4-AH, 60 g of HeLa nuclear extract or depleted HeLa nuclear extract, 30 ng of recombinant TFIIB, 1 unit of hexokinase (Sigma-Aldrich), and 2 mM glucose for 30 min at room temperature. The beads were pulled down again, and the supernatant was run on a denaturing SDS-acrylamide gel and Western blotted against TFIIB (C-18, Santa Cruz Biotechnology) and polymerase II (8WG16, Covance). Iron Cleavage Assay [28]—A total reaction mixture of 100 l containing 100 M DTT and 100 ng of TFIIB in 8 mM Hepes (pH 7.9) was preincubated at room temperature for 20 min. Samples were run on a denaturing SDS-acrylamide gel and Western blotted against TFIIB (C-18)
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