REPRESSOR AND ANTIREPRESSOR PROTEINS OF BACTERIOPHAGES

位点特异性DNA结合蛋白在调控中起重要作用 转录; DNA的复制和修复;噬菌体和病毒DNA 包装;位点特异性重组和转座，以及 限制-修改。 现代分子生物学的目标之一是 来从分子层面理解这些过程。 的真正理解 DNA识别的技术将允许设计蛋白质， 细胞或病毒DNA中的特定序列。 如果这些蛋白质可以 靶向适当的细胞，它们可能被用来调节基因 表达或病毒生长，因此在治疗 疾病 本提案中所述研究的目的是了解 阻遏物序列与结构的关系 蛋白质及其DNA识别和基因调控活性。作为 实验系统中，三种噬菌体阻遏物，P22 Arc，P22 Mnt， 将研究噬菌体λ阻遏物的N-末端结构域。 Arc和Mnt 是一种小型的同源蛋白质， 用于DNA识别的β-带状基序。 λ阻遏物是一种 螺旋-转角-螺旋DNA结合蛋白 这些系统中的每一个都很容易 可通过蛋白质和核酸的组合进行研究 化学，分子遗传学分析，动力学和平衡研究， 和X射线晶体学，从而使有意义的结构-功能 问题研究 蛋白质-DNA复合物的X射线晶体学研究将是 结合生物化学研究的影响， 取代突变对DNA自由能和特异性的影响 识别，以确定显着的特异性， DNA结合的变化可由单个氨基酸的变化引起。 的 Arc阻止或减缓RNA异构化的机制 转录过程中的聚合酶也将通过组合 生物化学和遗传学研究。 最佳的DNA结合位点是 在三种不同的结构背景下确定弧，以确定 如果四级结构限制了理想化的DNA接触，反之亦然。

Site specific DNA binding proteins play important roles in the regulation of transcription; the replication and repair of DNA; phage and viral DNA packaging; site-specific recombination and transposition, and restriction-modification. One of the goals of modem molecular biology is to understand these processes in molecular detail. A true understanding of DNA recognition would allow the design of proteins that could bind to specific sequences in cellular or viral DNA. If such proteins could be targeted to appropriate cells, they might be used to modulate gene expression or viral growth and thus be valuable in the treatment of disease. The objective of the research described in this proposal is to understand the relationship between the sequences and structures of repressor proteins and their DNA recognition and gene regulatory activities . As experimental systems, three phage repressors, P22 Arc, P22 Mnt, and the N-terminal domain of phage lambda repressor will be studied. Arc and Mnt are small, homologous proteins that use the recently discovered beta-ribbon motif for DNA recognition. Lambda repressor is a helix-turn-helix DNA binding protein. Each of these systems is readily accessible to study by a combination of protein and nucleic acid chemistry, molecular genetics analysis, kinetic and equilibrium studies, and X-ray crystallography, thereby allowing meaningful structure-function studies. X-ray crystallographic studies of protein-DNA complexes will be combined with biochemical studies of the effects of side chain substitution mutations on the free energy and specificity of DNA recognition to determine the mechanisms by which remarkable specificity changes in DNA binding can be caused by single amino acid changes. The mechanism by which Arc prevents or slows the isomerization of RNA polymerase during transcription will also be addressed by a combination of biochemical and genetic studies. Optimal DNA binding sites will be determined for Arc in three different structural backgrounds to determine if quaternary structure constrains idealized DNA contacts and vice versa.