Open Access Thesis
Date of Award
Philip R. Cunningham
X-ray crystallography of the prokaryotic 30S ribosomal subunit revealed a myriad of complex RNA-RNA, RNA-protein, and protein-protein interactions. Among these are several phylogenetically conserved RNA pseudoknots. Pseudoknots are structurally and functionally diverse RNA secondary structures. They are generally formed by two short complimentary sequences separated by many bases of single stranded regions or loops. These relatively simple folds are often yield complex structures that are key components of functionally important conformational changes in RNA structure. One such pseudoknot is located in the central domain of the 16S rRNA.
The central domain pseudoknot is formed by Watson-Crick base pairing between G570-C866 and U571-A865. Previous studies by other groups show that formation of this pseudoknot is critical for ribosome function. To examine the role this pseudoknot in ribosome function, we constructed and assayed all 255 possible mutations at these four residues. Our data show that disruption of base pairing between positions 570-866 reduces ribosome function by approximately 50% and that mutations that disrupt pairing between 571-865 completely block protein synthesis.
Ribosomal proteins S8, S11 and S5 have binding sites near the central pseudoknot. To determine if mutations in the pseudoknot affect the binding of these proteins, the genes for S8, S11 and S5 were cloned and coexpressed with representative mutations at each of the sites in the pseudoknot. No complementation was observed in any of the mutants tested, indicating that loss of function in the mutants is not due to reduction in binding of ribosomal proteins.
To determine the influence of thermodynamics on the activity of the mutants, each mutant was assayed at 25°C, 30°C, 37°C, and 40°C. Mutants with no measurable protein synthesis activity at 37°C were unaffected by changes in incubation temperatures. Mutants with partial activity, however, were slightly more active at 40°C but were strongly inhibited by incubation temperatures below 37°C. These data suggest that the central pseudoknot is dynamic and may facilitate the switch between two active conformations of rRNA. Mutations in the pseudoknot may therefore create thermodynamic minima that favor one conformation over the other. Homology modeling and ribosome profiles suggest that the mutations may affect ribosome association or one of the partial reactions during the protein synthesis process itself.
Jasinski-Bolak, Jenna Marie, "Investigation Of A 16s Rna Central Domain Pseudoknot" (2012). Wayne State University Theses. 210.