The long term objective of this research is to elucidate how the structure of RNA and DNA influences the function of these nucleic acids in biologically significant processes such as recognition, transcription, and replication. This goal will be accomplished by studying the structures of nucleic acids in model systems using nuclear magnetic resonance (NMR) spectroscopy. The structure of two RNA hairpins that contain mismatches and bulges will be determined. The mismatches include G U, G A, and tandem G A mismatches, all commonly found in a wide variety of different RNAs. The hairpins contain tandem G A mismatches in the RNA hairpins will be compared to previous results from DNA. One of the hairpins also contains a bulged base, another common structural motif in RNAs that has been implicated in RNA recognition by proteins. The hairpins will be isotopically labeled with 13C and 15N to take advantage of the multidimensional multinuclear NMR techniques that have previously been applied to proteins. These studies will provide a three-dimensional view of these structural motifs in RNA and will provide a foundation for modeling other RNA structures. The biologically significant problem of why the transcription process terminates spontaneously at specific sequences, so called simple transcription termination, will be modeled with RNA and DNA oligomers. These studies are based on the """"""""transcription bubble paradigm"""""""" and sequences from T7 and T3 bacteriophage will be used as model terminators. The terminator hairpin, an RNA/DNA hybrid, and a mixture of the """"""""nascent"""""""" RNA strand and the DNA template will be characterized using NMR spectroscopy. The proposed equilibrium between hybrid and hairpin formation will also be investigated. The RNA strands in these studies will be isotopically labeled with 13C and 15N allowing for selective observation of the RNA versus the DNA strands. These studies will provide structural, thermodynamic and kinetic information that will ultimately contribute to understanding the regulation of gene expression. Two DNA duplexes that contains a bulged quanosine at the 3-or the 5-end of an A T tract will be structurally characterized using NMR. The two duplexes will test the influence of the A T tract on the conformation of the bulged base. These results, combined with previous results, will examine the effect of sequence on the conformation of a single-base bulge in DNA and will further elucidate the process of frameshift mutagenesis.
