Ribonucleases (RNases) play a central role in a number of vital RNA cellular processes in all living cells. One of these processes is mRNA degradation, which is an important mechanism for post-transcriptional control of gene expression. RNases are also required for maturation and turnover of structural RNAs. E. coli has served as a model system for understanding the role of ribonucleases in cellular RNA metabolism, and eight distinct exoribonucleases have been identified in this bacterium. Of these, three (RNase T, Rnase D, and oligoribonuclease) are members of a larger exonuclease superfamily that includes the proof-reading domains of DNA polymerases. These three proteins share similar sequence motifs and have been dubbed the DEDD family exoribonucleases. However, functionally these exoribonuclease are quite distinct. We have initiated structural studies of this family of exoribonucleases, in collaboration with the laboratory of Dr. Murray Deutscher at the University of Miami, to structurally characterize these proteins. The structure of oligoribonuclease has been solved and we have diffraction quality crystals of RNase T. Specifically, we propose to: 1. Initiate detailed structure-function studies of oligoribonuclease to better understand its active site, metal requirements, and dimeric state. We also propose to look for differences between the prokaryotic and eukaryotic forms of this enzyme, by obtaining the structure of the human homologue of oligoribonuclease. 2. Optimize the crystals obtained for RNase T, and derive its atomic structure. 3. Determine the atomic structure of RNase D. 4. Understand the similarities and differences between these three enzymes in terms of substrate specificity, quarternary structure, metal requirements and catalytic mechanism, to better characterize this family of enzymes. The long term goals of this research are to understand the structures and mechanisms of action of all the exoribonucleases in a single organism (E. coli); these studies will complement a parallel study to completely determine and characterize the physiological role of all the exoribonucleases in E. coli, now underway in the Deutscher laboratory.
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