Abstract
The general transcription factor TFIIB plays a crucial role in selecting the transcription initiation site in yeast. We have analyzed the human homologs of TFIIB mutants that have previously been shown to affect transcription start site selection in the yeast Saccharomyces cerevisiae. Despite the distinct mechanisms of transcription start site selection observed in S. cerevisiae and humans, the role of TFIIB in this process is similar. However, unlike their yeast counterparts, the human mutants do not show a severe defect in supporting either basal transcription or transcription stimulated by an acidic activator in vitro. Transient transfection analysis revealed that, in addition to a role in transcription start site selection, human TFIIB residue Arg-66 performs a critical function in vivo that is bypassed in vitro. Furthermore, although correct transcription start site selection is dependent upon an arginine residue at position 66 in human TFIIB, innate function in vivo is determined by the charge of the residue alone. Our observations raise questions as to the evolutionary conservation of TFIIB and uncover an additional function for TFIIB that is required in vivo but can be bypassed in vitro.
Highlights
Transcription of a gene by RNA polymerase II1 requires the assembly of the general transcription factors (GTFs) at the promoter to form a preinitiation complex (PIC; reviewed in Ref. 1 and 2)
Human TFIIB Mutants E51R, R66E, and E51R/R66E Support Transcription in Vitro—The region of S. cerevisiae TFIIB that is involved in transcription start site selection is very highly conserved between species (Fig. 1A)
We have constructed single (E51R, R66E) and a double (E51R/R66E) point mutants of human TFIIB that correspond to the S. cerevisiae TFIIB mutants previously characterized by others to show defects in transcription start site selection [19]
Summary
Plasmids—The promoter DNA template G5E4T has been described previously [23]. G5ML contains nucleotides Ϫ50 to ϩ22 from the adenovirus major late promoter cloned downstream of 5 GAL4 sites in the vector pGEM3. The HeLa fraction containing RNA polymerase II, TFIIF, TFIIE, and TFIIH was purified as described previously [25]. Transcription assays using HeLa nuclear extract or TFIIB-depleted nuclear extracts were performed as described [13] using the amounts of recombinant TFIIB indicated in the figure legends. The reconstituted transcription assay was performed except it contained 20 l of the HeLa RNA pol II/TFIIF/TFIIE fraction, 20 ng of recombinant TBP, and the indicated amounts of TFIIB. 1 g of G5E4CAT (or G5MLCAT), 2 g of RSV-BxGALII, and 2 g of pCDNA3-TFIIB (or mutant) was diluted to 438 l with water, and 61 l of 2 M CaCl2 was added.
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