UBC Theses and Dissertations
A conformational study of 5-fluorouracil labeled Escherichia coli 5SrRNA Smith, James Lewis
¹⁹F-nmr spectroscopy and laser Raman spectroscopy are used to interpret conformational properties of 5SrRNA. When 5-fluorouracil is added to a medium containing actively growing E. coli cells it is incorporated into the 5SrRNA (FU-5SrRNA) of the. bacteria. The ¹⁹F-nmr spectra of FU-5SrRNA, at 94.1 MHz and 254 MHz, are presented. They cover a chemical shift range of approximately 8 p.p.m.. At 254 MHz the spectrum consists of 8 peaks and 2 shoulders, representing fluorine resonances from approximately twenty 5-fluorouracil residues in FU-5SrRNA. The most exposed residues have been assigned by comparison of the resonance frequency of the 5-fluoro-2'-deoxyuridine monophosphate monomer with that of the heat denatured FU-5SrRNA. The remainder of the fluorine resonances (about 70% of the total) are believed to be due to buried 5-fluorouracil residues. All the T₁ values of the individual peaks were approximately the same (between 0.3 and 0.4 seconds). This is in contrast to the T₁ value of the 5-fluoro-2'-deoxyuridine monomer which is approximately 5 seconds. A nuclear Overhauser enhancement experiment confirms that these residues are rigidly situated within the FU-5SrRNA molecule and that their molecular correlation time (τ) must be the same as the overall correlation time of the macromolecule. Two important results are obtained from laser Raman spectroscopic studies presented in this work. First, 5-fluoro-substitution affects certain vibrational properties of the uracil base. Second, from the comparison of N-5SrRNA spectra with FU-5SrRNA spectra, it appears evident that 5-fluorouracil substitution in 5SrRNA causes only minimal perturbation of the structure; thus, conclusions resulting from a study of FU-5SrRNA structure should be applicable to N-5SrRNA.
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