During the past year we have successfully colleced data on 10 protein and nucleic acid projects and have solved numerous structures, mostly complexes between proteins and wild-type or mutant nueleic acid substrates. We have obrtained a 3 _ resolution electron density map of T7 RNA polymerase complexed with a transcriptional inhibitor, lysozyme,and are proceeding to interpret this map. The higher resolution afforded by synchrotron data has allowed us to study glutaminyl-tRNA synthetase complexed with several tRNAs that were mutated in recognition elements both in the anticodon loop and in the acceptor stem. These structures have been refined and are providing insights into the mechanism of recognition. A superb 5 _ resolution electron density map of HIV reverse transcriptase complexed with a pseudoknot RNA inhibitor, identified by a SELEX procedure, has been obtained. Though at low resolution, the pseudoknot RNA can be clearly fitted to the map and lies between the polymerase active site and the RNase H active site interacting with both p51 and p66. The structure of Taq DNA polymerase complexed with duplex DNA at the polymerase active site has been solved and refined at 3 _ resolution. For the first time this structure gives us insight as to how duplex DNA binds to this DNA polymerse. Synchrotron radiation allowed us to extend the crystal structure of the CAP transcription activator complexed with 46 base-pairs of DNA to 2.2 _ resolution, a significant improvement from the previous structure which was at 3.0 _. In this new structure we have identified a second cyclic AMP binding site located in the DNA bidning domain. Since the resolution of Klenow fragment complexed with single stranded DNA can now be extended from 2.6 _ resolution to 2.1 _ resolution, we have been able to conduct a detailed study of this enzyme complexed with various phosphothiolate DNAs and various metal ions. High resolution refinement of these structures is providing a solid foundation for the two-metal-ion mechanism of catalysis. Data on other systems that have not yet immediately led to structure determinations but have been valuable for subsequent studies include data from a 56 nucleotide fragment of 5S RNA, a ROP RNA duplex, a fragment of the Rag I protein which is involved in immunoglobulin recombination, and the GIN recombinase complexed with DNA.
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