We propose to study the three-dimensional structure of DNA and RNA in unusual conformational states in order to uncover aspects which may be important to understanding their biological activities. The major experimental tool is single crystal x-ray diffraction analysis. Studies will be carried out on a Z-DNA, the left-handed conformer of the double helix. Experiments will be undertaken to discover the structure of the junction between right-handed B-DNA and left-handed Z-DNA. In Z-DNA, the nucleotides alternate in anti and syn conformations. We will try to find the three-dimensional structure at the junction between two segments of Z-DNA which are out of phase with each other relative to syn and anti conformation. The structure of ions that may act to stabilize Z-DNA will also be studied. Z-DNA is stabilized by polymers containing arginine residues. The structural basis of this will be addressed through co-crystallization of Z-DNA and arginine containing peptides. Z-DNA binding proteins have been isolated from E. coli and three of them have been cloned. We propose to study the three-dimensional structure of these proteins bound to fragments of Z-DNA, in order to uncover the structural motifs in proteins used in Z-DNA recognition. The biological role of Z-DNA is closely bound to our understanding of Z-DNA binding proteins. DNA can also adopt other alternative conformations in homopurine regions. These sequences are found in 5' flanking regions and may play a role in regulating gene expression. The structure of these segments will be studied. Segments of DNA containing alternations of thymine and adenine residues are also known to adopt an alternative conformation. Oligonucleotides will be synthesized and crystallized in an attempt to uncover the structure of these alternative conformations. The 5' end of eukaryotic mRNA contains a cap structure with m7GpppN. Variants of this will be made in order to discover whether the mRNA cap has a unique conformation. In addition, attempts will be made to co-crystallize these mRNA cap structures with a purified cap binding protein. These proteins act to remove secondary structure in mRNA and also facilitate messenger splicing activities. The structural basis of this will be studied by determining the three-dimensional structure of this complex.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Method to Extend Research in Time (MERIT) Award (R37)
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Massachusetts Institute of Technology
Schools of Arts and Sciences
United States
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