Dabas Preeti, Sweta Kumari, Ekka Mary, Sharma Nimisha
University School of Biotechnology, G.G.S. Indraprastha University, Sector16C, Dwarka, New Delhi 110078, India.
CSIR-Institute of Genomics and Integrative Biology, Mathura Road, Opp. Sukhdev Vihar Bus Depot, New Delhi, Delhi 110025, India.
Gene. 2018 Jan 30;641:117-128. doi: 10.1016/j.gene.2017.10.031. Epub 2017 Oct 13.
EAF (ELL Associated Factor) proteins interact with the transcription elongation factor, ELL (Eleven nineteen Lysine rich Leukemia) and enhance its ability to stimulate RNA polymerase II-mediated transcriptional elongation in vitro. Schizosaccharomyces pombe contains a single homolog of EAF (SpEAF), which is not essential for survival of S. pombe in contrast to its essential higher eukaryotic homologs. The physiological role of SpEAF is not well understood. In this study, we show that S. pombe EAF is important in regulating growth of S. pombe cells during normal growth conditions. Moreover, SpEAF is also essential for survival under conditions of DNA damage, while its deletion does not affect growth under environmental stress conditions. Our in vivo structure-function studies further demonstrate that while both the amino and carboxyl terminal domains of SpEAF possess the potential to activate transcription, only the amino terminal domain of SpEAF is involved in interaction with the S. pombe ELL protein. The carboxyl-terminus of SpEAF is required for rescue of the growth defect under normal and DNA damaging conditions that is associated with the absence of SpEAF. Using bioinformatics and circular dichroism spectroscopy, we show that the carboxyl-terminus of SpEAF has a disordered conformation. Furthermore, addition of trifluoroethanol triggered its transition from a disordered to α-helical conformation. Taken together, the results presented here identify novel structural and functional features of SpEAF protein, providing insights into how EAF proteins may enforce transcriptional control of gene expression.
EAF（ELL相关因子）蛋白与转录延伸因子ELL（富含11 - 19赖氨酸的白血病蛋白）相互作用，并在体外增强其刺激RNA聚合酶II介导的转录延伸的能力。粟酒裂殖酵母含有EAF的单一同源物（SpEAF），与其在高等真核生物中必需的同源物不同，SpEAF对粟酒裂殖酵母的存活并非必需。SpEAF的生理作用尚不清楚。在本研究中，我们表明粟酒裂殖酵母EAF在正常生长条件下调节粟酒裂殖酵母细胞的生长中很重要。此外，SpEAF在DNA损伤条件下的存活中也是必需的，而其缺失并不影响在环境应激条件下的生长。我们的体内结构 - 功能研究进一步表明，虽然SpEAF的氨基末端和羧基末端结构域都具有激活转录的潜力，但只有SpEAF的氨基末端结构域参与与粟酒裂殖酵母ELL蛋白的相互作用。SpEAF的羧基末端是在正常和DNA损伤条件下挽救与SpEAF缺失相关的生长缺陷所必需的。使用生物信息学和圆二色光谱法，我们表明SpEAF的羧基末端具有无序构象。此外，添加三氟乙醇会触发其从无序构象转变为α - 螺旋构象。综上所述，本文给出的结果确定了SpEAF蛋白的新结构和功能特征，为EAF蛋白如何加强基因表达的转录控制提供了见解。
