9317196 Davis NMR spectroscopy will be used to determine the structure of RNA oligonucleotides corresponding to structural domains in tRNAs. Synthetic oligonucleotides corresponding to the anticodon stem- loop region and the dihydrouridine stem-loop region will be synthesized using synthetic phosphoramidite methods. The naturally occurring modified nucleosides pseudouridine, 2'-O- methylcytidine, 2-methylthio-N6-isopentenyladenosine, 1- methylguanosine, 5-methylaminomethyl-2-thiouridine, and dihydrouridine will be incorporated into RNA oligonucleotides. The structures for the modified oligonucleotides will be determined and compared to the structures of the unmodified analogs. Structural changes induced by RNA modification will be correlated to changes in the stability, both in terms of overall structural integrity, and local conformational dynamics. The effect of divalent metal ion coordination on the structure and stability will be determined for unmodified and modified oligonucleotides. In cases where high affinity magnesium coordination sites are indicated by KD measurements, the specific metal ion bindings sites will be determined in subsequent high- resolution structural studies. Traditional 2D NMR methods along with newer, homonuclear 3D NMR experiments will be used to assign RNA resonances and to derive structural information. Distance geometry and molecular dynamics calculational methods will be used to convert NMR structural constraints into well-defined 3D structures. The structural data obtained for tRNA oligonucleotide analogs will be used to explain the role of modified nucleosides where these particular nucleosides have been implicated in tRNA function. %%% Transfer RNA is a unique biological molecule that performs an integral role in translating the genetic message contained in DNA. The DNA code in a gene is ultimately used to construct specific proteins, tRNAs specific for each of the amino acids that go into a protein are part of th e ribosomal protein synthesis machinery. The three-dimensional structure of each amino acid specific tRNA is unique and this structural diversity is the basis for assuring that the correct amino acid is inserted into the protein sequence in response to the appropriate message from the DNA. tRNAs are composed of the ribonucleosides adenosine, uridine, cytidine, and guanosine as well as an enormous array of chemically "unusual" nucleosides that are derivatives of the four common ribonucleosides. These "modified" nucleosides appear to play a critical role in defining the structures of tRNAs. The modified nucleosides are important in assuring that tRNAs function correctly and also in discriminating between tRNAs that code uniquely for each of the common amino acids. The goal of this proposal is to further the understanding of the function of modified nucleosides in tRNA. The way in which chemical modification influences structure is likely to be critical to the correct function of tRNAs. We will use a powerful tool for elucidating the structure of tRNA, NMR spectroscopy, to produce three-dimensional structures of modified RNAs. The information derived from these studies will aid in understanding the complex function of a biological macromolecule that is central to all life. ***
