SPECTROSCOPY OF HEAVY ATOM PERTURBED BIOPOLYMERS

重原子扰动生物聚合物的光谱

• 批准号: 3249967
• 负责人: AUGUST H MAKI
• 金额: $ 12.87万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 1981
• 资助国家: 美国
• 起止时间: 1981-01-01 至 1987-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3249967
• 关键词: DNA; DNA directed RNA polymerase; Escherichia coli; RNA; bacterial virus; chemical binding; circular DNA; gel electrophoresis; genetic manipulation; genetic regulation; heavy metals; intermolecular interaction; lac operon; mercury; metal complex; methylmercury; nuclear magnetic resonance spectroscopy; nucleic acid chemical synthesis; nucleic acid sequence; nucleotides; operon; polymers; polynucleotides; silver; triplet state

In the long term, the objective of this research program is to acquire detailed knowledge of protein-nucleic acid interactions at a molecular level. Use will be made of optical detection of triplet state magnetic resonance (ODMR) methodology to investigate both sequence-specific and sequence-non-specific polynucleotide-protein complexes. We will take advantage of the external heavy atom effect which operates at extremely short range to probe for contact interactions between aromatic residues of nucleic acid binding proteins and heavy atom-derivatized nucleic acid bases in these complexes using ODMR methodologies. We will test for the generality of a model suggested by previous work of a sequence-non-specific complex which requires that the nucleic acid bases are buried in interior hydrophobic regions of the single-strand binding protein (SSBP), by making ODMR measurements on complexes of single stranded heavy atom-derivatized polynucleotides and gene 32 (T4), gene 5 (fd), and SSBP (T7). In order to produce specific sequences of DNA containing heavy atom derivatization at specific sites, we will utilize the phage M13mp8 to a large extent. It has a single stranded closed circular DNA containing a cloned segment from the E. coli lac operon. The latter will be excised from the RF DNA using AvaI, derivatized with Hg, and complexed with the specific binding proteins, RNA polymerase, CAP, and lac repressor from E. coli. The complexes will be examined for heavy atom effects using ODMR. In addition, the M13mp8 single stranded DNA will be used as a template for in vitro DNA synthesis with heavy atom-derivatized nucleotides incorporated into the synthesized strand. The lac operon will be excised using AvaI, and complexes with E. coli RNA polymerase, CAP, and lac repressor will be investigated. Also, restriction endonuclease recognition sequences will be excised from the polylinker region of M13mp8, and complexes will be formed with the appropriate endonucleases, and studied by ODMR spectroscopy. In similar experiments, the A1, A2, and A3 early promoters of T7 bacteriophage will be obtained by growth of the phage and scission of the terminal DNA segment of 815 bp containing these promoters using AluI. Further digestion of the terminal fragment with HpaII will produce smaller fragments containing the individual promoters. Heavy atom derivatives will be made, and complexes of RNA polymerase with the individual promoters will be studied using ODMR. In the area of structurally relevant protein-nucleic interactions, we will use ODMR to study metal ion (Ag+, Hg2+, CH3Hg+) binding to the DNA of various filamentous phages.

从长远来看，这项研究计划的目标是获得 对蛋白质-核酸相互作用的详细了解 水平。将使用三重态磁的光学检测 共振(ODMR)方法研究序列特异性和 序列-非特异的多核苷酸-蛋白质复合体。我们会带上 外部重原子效应的优势，它工作在极高的 近距离探测芳香族残基之间的接触相互作用 核酸结合蛋白和重原子衍生的核酸碱基 在这些复合体中使用ODMR方法。我们将测试 由序列的先前工作提出的模型的一般性--非特定 需要将核酸碱基埋在内部的复合体 单链结合蛋白(SSBP)的疏水区，通过制造 单链重原子衍生化配合物的ODMR测量 多核苷酸和基因32(T4)、基因5(Fd)和SSBP(T7)。为了 产生含有重原子衍生化的特定DNA序列 对于特定的位点，我们将在很大程度上利用噬菌体M13mp8。它有 含有克隆片段的单链闭合环状DNA 大肠杆菌乳胶操纵子。后者将使用Avai从RF DNA中切除， 用汞衍生，并与特定结合蛋白RNA络合 来自大肠杆菌的聚合酶，CAP和乳胶抑制物。这些综合体将是 使用ODMR检查了重原子效应。此外，M13mp8单曲 链DNA将被用作体外DNA合成的模板 重原子衍生核苷酸掺入合成的 斯特兰德。用Avai切除乳胶操纵子，并与E. 将对Coli RNA聚合酶、CAP和Lac阻遏物进行研究。另外， 限制性内切酶识别序列将从 M13mp8的多连接区，并与M13mp8形成复合物 合适的内切酶，并用ODMR波谱进行研究。在类似的 实验中，T7噬菌体的A1、A2和A3早期启动子将被 通过噬菌体的生长和末端DNA片段的断裂获得 含有使用Alui的这些启动子的815个核苷酸。进一步消化 带有HpaII的末端片段将产生包含 个人发起人。重原子衍生品将被制造出来，而络合物 RNA聚合酶与单个启动子的关系将用 ODMR。在结构相关的蛋白质-核相互作用领域， 我们将使用ODMR来研究金属离子(Ag+，Hg2+，CH3Hg+)与DNA的结合 各种丝状噬菌体。