SPECTROSCOPY OF HEAVY ATOM PERTURBED BIOPOLYMERS

重原子扰动生物聚合物的光谱

基本信息

批准号：
3249972
负责人：
AUGUST H MAKI
金额：
$ 8.48万
依托单位：
UNIVERSITY OF CALIFORNIA AT DAVIS
依托单位国家：
美国
项目类别：
财政年份：
1981
资助国家：
美国
起止时间：
1981-01-01 至 1988-06-30
项目状态：
已结题

项目摘要

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，该研究计划的目标是获得蛋白质-核酸分子相互作用的详细知识等级。将利用光学检测三重态磁共振（ODMR）方法来研究序列特异性和序列非特异性多核苷酸-蛋白质复合物。我们将采取利用外部重原子效应的优势，该效应在极端条件下运行短距离探测芳香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使用 ODMR 方法对这些复合物进行分析。我们将测试序列非特异性的先前工作所建议的模型的一般性复合物需要核酸碱基埋在内部单链结合蛋白（SSBP）的疏水区域，通过使单链重原子衍生复合物的 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 序列在特定位点上，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环DNA 大肠杆菌乳糖操纵子。后者将使用 AvaI 从 RF DNA 中切除，用 Hg 衍生化，并与特异性结合蛋白 RNA 复合来自大肠杆菌的聚合酶、CAP 和 lac 阻遏蛋白。这些综合体将是使用 ODMR 检查重原子效应。此外，M13mp8单链状 DNA 将用作体外 DNA 合成的模板重原子衍生的核苷酸并入合成的股。将使用 AvaI 切除 lac 操纵子，并与大肠杆菌形成复合物。将研究大肠杆菌 RNA 聚合酶、CAP 和 lac 阻遏蛋白。还，限制性内切核酸酶识别序列将从 M13mp8 的多接头区域，并且将与形成复合物适当的核酸内切酶，并通过 ODMR 光谱进行研究。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 815 bp 包含使用 AluI 的这些启动子。进一步消化带有 HpaII 的末端片段会产生更小的片段，其中包含个人发起人。将制备重原子衍生物和络合物将使用以下方法研究 RNA 聚合酶与各个启动子的关系 ODMR。在结构相关的蛋白质-核相互作用领域，我们将使用 ODMR 研究金属离子（Ag+、Hg2+、CH3Hg+）与 DNA 的结合各种丝状噬菌体。