Abstract
Yeast TFIID comprises the TATA binding protein and 14 TBP-associated factors (TAF(II)s), nine of which contain histone-fold domains (HFDs). The C-terminal region of the TFIID-specific yTAF4 (yTAF(II)48) containing the HFD shares strong sequence similarity with Drosophila (d)TAF4 (dTAF(II)110) and human TAF4 (hTAF(II)135). A structure/function analysis of yTAF4 demonstrates that the HFD, a short conserved C-terminal domain (CCTD), and the region separating them are all required for yTAF4 function. Temperature-sensitive mutations in the yTAF4 HFD alpha2 helix or the CCTD can be suppressed upon overexpression of yTAF12 (yTAF(II)68). Moreover, coexpression in Escherichia coli indicates direct yTAF4-yTAF12 heterodimerization optimally requires both the yTAF4 HFD and CCTD. The x-ray crystal structure of the orthologous hTAF4-hTAF12 histone-like heterodimer indicates that the alpha3 region within the predicted TAF4 HFD is unstructured and does not correspond to the bona fide alpha3 helix. Our functional and biochemical analysis of yTAF4, rather provides strong evidence that the HFD alpha3 helix of the TAF4 family lies within the CCTD. These results reveal an unexpected and novel HFD organization in which the alpha3 helix is separated from the alpha2 helix by an extended loop containing a conserved functional domain.
Highlights
Accurate transcription initiation at protein-coding genes by RNA polymerase II requires the assembly of a multiprotein complex around the mRNA start site [1]
Sequence comparisons and structural studies indicated that TAF6 and TAF9 contain histone-fold domain (HFD) similar to those of core histones H4 and H3, which interact to form an H3-H4-like heterotetramer [27, 28]. hTAF4 and hTAF12 heterodimerize via HFDs similar to those of H2A and H2B, respectively [29]
We demonstrate that the conserved Cterminal domain (CCTD) of yTAF4 interacts genetically with yTAF12, is broadly required for transcription in vivo, and is required for optimal heterodimerization with yTAF12 in vitro
Summary
Accurate transcription initiation at protein-coding genes by RNA polymerase II requires the assembly of a multiprotein complex around the mRNA start site [1]. We show that the HFD is essential, but not sufficient, for yTAF4 function, which requires the short highly conserved C-terminal domain (CCTD) and the intervening linker region.
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