We propose to solve the three-dimensional structure of a number of proteins that interact with the nucleic acids using x-ray diffraction analysis. We propose to work with systems which are involved in the recognition of different types of nucleic acid structures. Attempts will be made to solve the crystal structure of mutants of fragments of the E. coli alanyl tRNA synthetase with or without its cognate tRNA attached. The synthetases play a key role in insuring the fidelity of the transfer of information from nucleic acids to proteins but the molecular basis of their recognition is unknown. In another system we will investigate the structure of a protein which binds to specific DNA sequences. We propose to solve the structure of the SP01 bacteriophage encoded protein, transfer factor 1 (TF1) which binds to particular sequences of bacteriophage DNA. TF1 is closely related to the E. coli encoded Hu protein which binds to any double helical DNA, independent of sequence. However, bacteriophage SP01 encoded TF1 binds DNA in a sequence specific manner. We wish to know how two related proteins, Hu and TF1 bind to DNA in sequence independent and dependent modes. Site-specific mutagenesis can be used to develop variants of the proteins. Monoclonal antibodies have been raised against left-handed Z- DNA which was formed with a sequence containing alternations of cytosine and guanine residues. Similar antibodies are found in the autoimmune disease systemic lupus erythematosis. The antibodies are of two types, one is specific for this sequence, and the other appears to bind to the left-handed Z-DNA conformation in a manner independent of sequence. We will try to co-crystallize Z-DNA with Fab fragments of these monoclonal antibodies. Solution of this structure will provide us with information about the molecular mechanisms which are used to identify left-handed Z-DNA.
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