Feigerle Jordan T, Weil P Anthony
From the Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0615.
From the Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0615
J Biol Chem. 2016 Oct 21;291(43):22721-22740. doi: 10.1074/jbc.M116.751107. Epub 2016 Sep 1.
The evolutionarily conserved RNA polymerase II transcription factor D (TFIID) complex is composed of TATA box-binding protein (TBP) and 13 TBP-associated factors (Tafs). The mechanisms by which many Taf subunits contribute to the essential function of TFIID are only poorly understood. To address this gap in knowledge, we present the results of a molecular genetic dissection of the TFIID subunit Taf2. Through systematic site-directed mutagenesis, we have discovered 12 taf2 temperature-sensitive (ts) alleles. Two of these alleles display growth defects that can be strongly suppressed by overexpression of the yeast-specific TFIID subunit TAF14 but not by overexpression of any other TFIID subunit. In Saccharomyces cerevisiae, Taf14 is also a constituent of six other transcription-related complexes, making interpretation of its role in each of these complexes difficult. Although Taf14 is not conserved as a TFIID subunit in metazoans, it is conserved through its chromatin-binding YEATS domain. Based on the Taf2-Taf14 genetic interaction, we demonstrate that Taf2 and Taf14 directly interact and mapped the Taf2-Taf14 interaction domains. We used this information to identify a Taf2 separation-of-function variant (Taf2-ΔC). Although Taf2-ΔC no longer interacts with Taf14 in vivo or in vitro, it stably incorporates into the TFIID complex. In addition, purified Taf2-ΔC mutant TFIID is devoid of Taf14, making this variant a powerful reagent for determining the role of Taf14 in TFIID function. Furthermore, we characterized the mechanism through which Taf14 suppresses taf2 alleles, shedding light on how Taf2-Taf14 interaction contributes to TFIID complex organization and identifying a potential role for Taf14 in mediating TFIID-chromatin interactions.
进化上保守的RNA聚合酶II转录因子D（TFIID）复合物由TATA盒结合蛋白（TBP）和13个TBP相关因子（Tafs）组成。许多Taf亚基对TFIID基本功能的贡献机制目前了解甚少。为了填补这一知识空白，我们展示了对TFIID亚基Taf2进行分子遗传学剖析的结果。通过系统的定点诱变，我们发现了12个taf2温度敏感（ts）等位基因。其中两个等位基因表现出生长缺陷，这种缺陷可被酵母特异性TFIID亚基TAF14的过表达强烈抑制，但不能被任何其他TFIID亚基的过表达抑制。在酿酒酵母中，Taf14也是其他六种转录相关复合物的组成部分，这使得解释其在每种复合物中的作用变得困难。虽然Taf14在后生动物中不是作为TFIID亚基保守的，但它通过其染色质结合YEATS结构域保守。基于Taf2 - Taf14的遗传相互作用，我们证明Taf2和Taf14直接相互作用并绘制了Taf2 - Taf14相互作用结构域。我们利用这些信息鉴定了一个Taf2功能分离变体（Taf2 - ΔC）。虽然Taf2 - ΔC在体内或体外不再与Taf14相互作用，但它稳定地整合到TFIID复合物中。此外，纯化的Taf2 - ΔC突变TFIID不含Taf14，这使得该变体成为确定Taf14在TFIID功能中作用的有力试剂。此外，我们表征了Taf14抑制taf2等位基因的机制，阐明了Taf2 - Taf14相互作用如何有助于TFIID复合物组织，并确定了Taf14在介导TFIID - 染色质相互作用中的潜在作用。
