SPECTROSCOPY OF HEAVY ATOM PERTURBED BIOPOLYMERS

从长远来看，这个研究项目的目标是获得

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.