Molecular aspects of TAL effector-DNA binding

植物致病性黄单胞菌的天然TAL效应物（TALEs）在植物细胞中起转录因子的作用。它们采用重复的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），它们指定了哪个DNA碱基被结合。这种独特而新颖的dna结合模式使设计的TALE衍生物能够用于靶向表达，遗传电路控制和chirurgic基因组编辑，为基础科学和生物技术提供了深远的可能性。在这个提议中，我们的目的是澄清几个分子参数的dna - tale结合。首先，我们将分析新的RVD特异性及其效率，强RVD所需的最小数量和确切位置，以及靶结合序列中不匹配的影响。为了进一步了解这些规律的分子基础，我们将使用表面等离子体共振和微尺度热泳术来量化具有不同rvd的人工故事的DNA结合。最后，将分析不寻常的天然TALE重复序列和来自TALE同源序列的重复序列进行DNA结合，以了解如何在不失去DNA结合活性的情况下修饰TALE重复序列的氨基酸主干。这些研究将阐明tale - dna相互作用的规则，并有助于从总体上理解重复蛋白与核酸的结合。

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.