SPECTROSCOPY OF HEAVY ATOM PERTURBED BIOPOLYMERS

从长远来看，这项研究计划的目标是获得 分子水平上蛋白质-核酸相互作用的详细知识 水平 将使用三重态磁性的光学检测。 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 外部重原子效应的优势， 短程探针用于芳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 在这些复合物中使用ODMR方法。 我们将测试 一个模型的一般性建议由以前的工作序列非特异性 复合物，其要求核酸碱基被埋在内部， 单链结合蛋白（SSBP）的疏水区，通过使 单链重原子衍生物复合物的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的DNA的特定序列， 特异性位点，我们将在很大程度上利用噬菌体M13 mp8。 它有 单链闭合环状DNA，其含有来自 E.大肠杆菌乳糖操纵子。 后者将使用Avlantine从RF DNA中切除， 汞衍生，并与特异性结合蛋白，RNA复合 聚合酶、CAP和来自E.杆菌 这些复合体将 使用ODMR检查重原子效应。 此外，M13 mp8单曲 链DNA将用作体外DNA合成的模板， 重原子衍生的核苷酸掺入合成的 搁浅。 将使用Avlantine切除lac操纵子，并与E. coli RNA聚合酶、CAP和lac阻遏物。 还有， 限制性内切核酸酶识别序列将从所述细胞中切除。 M13 mp8的多接头区域，并且将与M13 mp8的多接头区域形成复合物。 合适的核酸内切酶，并通过ODMR光谱学研究。 以类似 在实验中，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噬菌体的末端DNA片段的断裂获得， 815 bp，包含这些启动子。 进一步消化 末端片段与HpaII的结合将产生更小的片段， 个人推广者。 重原子衍生物将被制造出来， RNA聚合酶与单个启动子的关系将使用 ODMR。 在结构相关的蛋白质-核酸相互作用领域， 我们将利用ODMR研究金属离子（Ag+，Hg ~（2+），CH_3 Hg ~+）与DNA的结合 各种各样的丝状真菌。

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.