Open Access Dissertation
Date of Award
The structures of two functional mutants, the UC (G690U, U697C) and the QM mutants (G690A, G693C, A695C, U697A) of the 690 hairpin of E. coli 16S ribosomal RNA were determined by NMR. The UC mutant and the QM mutant with high biological function are able to fold into structures that are isomorphous with the wild-type 690 hairpin sequence. The structural comparisons among the functional mutants and the wild-type provides structural validation for previously identified specific functional groups that are crucial for maintaining function of the 690 hairpin. The key groups for maintaining the structure and function of the 690 loop include the formation of a "U-turn" at position 692, mismatch pairs involving residues 691-696 and 690-697, the placement of a hydrogen bond acceptor atom in the minor groove at 697, and continuous stacking of five bases at the 3'-terminus of the hairpin.
The 970 hairpin of E. coli 16S rRNA contains two modified nucleotides, m2G966 and m5C967. The two modified nucleotides in the 970 loop are involved in some important biological functions, for example, the m2G966 of the 970 hairpin interact with C34 of P-site bounded tRNA in the crystal structure. The structural role of the modifications, however, is not obvious in the published crystal structures (2AVY and 1J5E), and the 970 loop structures in the two crystal structures have some differences, possibly because that the 970 loop hairpin can form multiple functionally important conformations. We determined NMR structures of the 970 hairpin with and without modifications to identify the functional roles of the modifications on the structures of the 970 loop. And the experimental results indicate that the chemical modifications in the 970 loop cause the structural differences between the NMR structures of the 970 loop with and without modifications, for example, the flipping G971. And the modified nucleotides in the 970 loop may contribute to the structural stability of the 970 loop because methyl groups in the two modified residues 966 and 967 increase the stacking surface area of the triple-base stacking formed among 966, 967 and 967. In addition, the NMR structure of the modified-970 RNA support the mutation study results obtained in Dr. Cunningham's lab.
Liu, Yu, "Nmr Solution Structures Of Two Hairpins Of E. Coli 16s Rrna: The Effects Of Mutations And Chemical Modifications On Structure And Function Of Rrna" (2011). Wayne State University Dissertations. 174.