Abstract
General transcription factor TFIID is a cornerstone of RNA polymerase II transcription initiation in eukaryotic cells. How human TFIID—a megadalton-sized multiprotein complex composed of the TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs)—assembles into a functional transcription factor is poorly understood. Here we describe a heterotrimeric TFIID subcomplex consisting of the TAF2, TAF8 and TAF10 proteins, which assembles in the cytoplasm. Using native mass spectrometry, we define the interactions between the TAFs and uncover a central role for TAF8 in nucleating the complex. X-ray crystallography reveals a non-canonical arrangement of the TAF8–TAF10 histone fold domains. TAF2 binds to multiple motifs within the TAF8 C-terminal region, and these interactions dictate TAF2 incorporation into a core–TFIID complex that exists in the nucleus. Our results provide evidence for a stepwise assembly pathway of nuclear holo–TFIID, regulated by nuclear import of preformed cytoplasmic submodules.
Highlights
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The importance of this cooperative activity of TAF8 and TAF10 is supported by nuclear import assays, which showed that the transport of TAF10 from the cytoplasm to the nucleus depends on the nuclear localization signal (NLS) found at the carboxyl-terminal (C-terminal) end of TAF8
We demonstrate that the TAF2–TAF8 interaction is crucial for formation of the cytoplasmic TAF2–8–10 complex, and dictates incorporation of TAF2 into a physiological core–TFIID complex that exists in the nucleus
Summary
General rights This document is made available in accordance with publisher policies. TAF3, 4, 6, 8, 9, 10, 11, 12 and 13 contain histone fold domains (HFDs), which stabilize discrete heterodimers (TAF3–10, TAF4–12, TAF6–9, TAF8–10 and TAF11–13) (refs 22–25) Among these HFD pairs, the TAF8–10 heterodimer plays a key role in the TFIID assembly pathway, is critical for the integrity of holo–TFIID and fulfills essential functions in early embryonic development[6,8,26,27]. The same phenotype was found in TAF10-knockout mice strongly suggesting that TAF8 and TAF10 are both involved in controlling embryonic development at similar stages[6] The importance of this cooperative activity of TAF8 and TAF10 is supported by nuclear import assays, which showed that the transport of TAF10 from the cytoplasm to the nucleus depends on the nuclear localization signal (NLS) found at the carboxyl-terminal (C-terminal) end of TAF8 The importance of this cooperative activity of TAF8 and TAF10 is supported by nuclear import assays, which showed that the transport of TAF10 from the cytoplasm to the nucleus depends on the nuclear localization signal (NLS) found at the carboxyl-terminal (C-terminal) end of TAF8 (ref. 30)
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