Molecular aspects of TAL effector-DNA binding

植物致病黄单胞菌的天然TAL效应(TALE)。细菌在植物细胞中扮演转录因子的角色。它们使用了一个重复的DNA结合域，具有可预测和可编程的特异性。每个重复序列有两个相邻的氨基酸，称为重复可变双残基(RVDS)，指定与哪个DNA碱基结合。这种独特而新颖的DNA结合模式使构建用于靶向表达、基因电路控制和化学基因组编辑的设计者故事衍生品成为可能，为基础科学和生物技术带来了深远的可能性。在这项建议中，我们旨在澄清TALL-DNA结合的几个分子参数。首先，我们将分析新的RVD特异性及其效率，强RVDS所需的最小数量和准确位置，以及靶结合序列中错配的影响。为了进一步了解这些规则的分子基础，将使用表面等离子体共振和微尺度热电泳法对不同RVDS的人工故事的DNA结合进行量化。最后，将分析不寻常的自然TALL重复和来自TALL同源物的重复DNA结合，以了解如何在不失去DNA结合活性的情况下对TALL重复序列的氨基酸骨架进行修饰。这些研究将阐明TALL-DNA-相互作用的基本规则，并有助于全面了解重复蛋白质与核酸的结合。

Natural TAL effectors (TALEs) from plant-pathogenic Xanthomonas spp. bacteria act as transcription factors in plant cells. They employ a repetitive DNA-binding domain with predictable and programmable specificity. Two neighboring amino acids per repeat, termed repeat variable diresidues (RVDs), specify which DNA base is bound. This unique and novel DNA-binding mode has enabled construction of designer TALE derivatives for targeted expression, control of genetic circuits, and chirurgic genome editing with far reaching possibilities for basic science and biotechnology. In this proposal we aim to clarify several molecular parameters of TALE-DNA binding. First, we will analyze novel RVD specificities and their efficiencies, the minimal number and exact position required for strong RVDs, and the impact of mismatches in the target binding sequence. To further understand the molecular basis of these rules, DNA binding of artificial TALEs with different RVDs will be quantified using surface plasmon resonance and microscale thermophoresis. Finally, unusual natural TALE repeats and repeats from TALE homologs will be analyzed for DNA binding to understand how the amino acid backbone of TALE repeats can be modified without losing DNA-binding activity. These studies will clarify rules underlying TALE-DNA-interaction, and help to generally understand binding of repetitive proteins to nucleic acids.