酵母一般转录因子TFIIIC对tRNA促进体参与的结构和运动见解
Wolfram Seifert-Dávila1, Anastasiia Chaban1,2, Florence Baudin1
1Structural and Computational Biology Unit, European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, 69117 Heidelberg, Germany.
Nucleic acids research
|December 10, 2024
概括
研究人员使用冷电子显微镜 (cryo-EM) 和单分子光共振能量转移 (smFRET) 揭示了转录因子IIIC (TFIIIC) 如何结合转移RNA (tRNA) 基因促进体. 这提供了对转录启动的结构和运动见解.
科学领域:
- 分子生物学分子生物学
- 结构生物学 结构生物学
- 生物化学 生物化学
背景情况:
- 转移RNA (tRNA) 基因由RNA聚合酶III (Pol III) 转录对于细胞功能至关重要.
- 这一过程需要通用转录因子IIIC (TFIIIC),该转录因子与II型基因促进体中的特定DNA基因 (A盒和B盒) 结合.
- 由于缺乏结构数据,TFIIIC与这些促进因素,特别是A-box的相互作用的确切机制尚不清楚.
研究的目的:
- 阐明酵母TFIIIC复合物识别和结合tRNA基因促进体内的A盒和B盒DNA基因的结构机制.
- 为了获得TFIIIC与tRNA基因促进者的实时动态洞察力.
- 了解不同TFIIIC子复合体 (τA和 τB) 在促进者识别和随后的其他转录因子招募中的作用.
主要方法:
- 使用单颗粒冷电子显微镜 (cryo-EM) 来确定与DNA结合的TFIIIC子综合体的高分辨率结构.
- 单分子光共振能量转移 (smFRET) 用于监测TFIIIC结合和构造变化的实时动力学.
- 进行了生物化学足迹实验,以评估TFIIIC特定组件和DNA元素在招聘过程中的作用.
主要成果:
- 与A盒和B盒DNA基因结合的tA和tB子复合体的高分辨率冷EM结构分别获得了3.7 Å和2.5 Å的分辨率.
- 低温电磁单颗粒测绘揭示了tA和tB子综合体的精确距离和相对方向,说明了在发起者上完全投入的TFIIIC综合体.
- smFRET实验表明,B盒相互作用主要决定TFIIIC在tRNA基因上的整体招募和停留时间.
- 足迹测定表明,在TFIIIC绑定II型促进者后,tA子综合体和A盒在招募TFIIIB和Pol III方面发挥着至关重要的作用.
结论:
- 该研究提供了TFIIIC引入tRNA基因促进体的第一个结构和动力特征,详细介绍了A盒和B盒识别的分子基础.
- TFIIIC绑定是一个多步骤的过程,其中B盒识别是初始稳定关联的关键,而A盒相互作用对于随后的核心转录机制的招募至关重要.
- 这些发现大大提高了我们对Pol III的tRNA基因转录启动机制的理解.
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