Open Access Thesis
Date of Award
Christine S. Chow
The importance of helix 69 (H69) of the large ribosomal subunit is emphasized by its location in the ribosome and key contacts with protein factors and tRNAs during different steps of protein synthesis. The presence of three highly conserved pseudouridine modifications in H69 has a significant impact on modulation of the ribosome conformations. Specific nucleotides of H69 may be necessary for the function of this helix-loop RNA, as some mutations are deleterious to bacterial growth. In this study, we investigated the effects of single nucleotide mutations in a 19-nucleotide H69 construct with and without pseudouridines on the RNA stability and conformation. A variety of experimental methods were employed, such as thermal melting, circular dichroism spectroscopy and 1D proton NMR spectroscopy. While all of the mutants have unique pH-dependent conformational changes, those changes didn't have an effect on the global stability of H69. Pseudouridines are involved in the modulation of conformations of each of the mutants. Nuclear magnetic resonance (NMR) spectra of mutants A1912G and A1919G also revealed altered conformations of H69 at different pH values as well as the importance of pseudouridine in regulating these changes. Lastly, the impact of these modifications on the ligand binding was examined.
Kharel, Daya Nidhi, "Biophysical Studies Of Mutant H69 Rna Constructs" (2014). Wayne State University Theses. 347.