Doctor of Philosophy (PhD)
The Saccharomyces cerevisiae ribosomal protein L30 is one of a unique group of proteins that bind to their own mRNA in a feedback regulation mechanism. L30 binds to a stem-loop-stem region in the RPL30 pre-mRNA which is called the kink-turn motif. This motif is found in several protein-binding RNAs in the ribosome, as well as in some small ribonucleoprotein particles such as components of the spliceosomal assembly. In order to study the interaction of L30 with its RPL30 kinkturn, several mutations have been made in L30 protein at key positions in the protein/ RNA interface. Phenylalanine 85 has been shown to provide a stabilizing stacking interaction with the purine-rich kink-turn loop in the RPL30 transcript.
Using past NMR and current crystallographic structural information about yeast L30 and phylogenetic comparison of L30 proteins in other species, single amino acid mutations at positions 84 and 85 in L30 protein were designed to study the effect of those changes on RNA/protein binding. Amino acids were selected for mutation to establish the necessity of an aromatic and hydrophobic residue in L30 position 85. The phenylalanine in position 85 was mutated to tryptophan, histidine, and isoleucine and the dissociation constant of each mutated L30 was compared to that of the wild type L30 using radioactive filter binding and band shift assays. To further study the position 85 region of L30, single alanine insertions at positions 84 and 86 and single amino acid mutations at position 84 such as proline, histidine and tyrosine were also made and assayed using filter binding and band shift experiments. In order of strongest binding to least, the position 85 mutants were histidine, tryptophan, and isoleucine, with Kd values of 4.3 nM, 10.5 nM, and 58.7 nM, respectively. Both alanine insertions exhibited decreased binding with Kd values for A84 and A86 being 58.2 nM and 170.4 nM, with the insertion at position 86 being the most disruptive. These results suggested that the F85 position needs to have an aromatic amino acid and the stacking interaction with guanine is quite specific.
The structural properties and stability of some of the mutants in differing solvent conditions compared to the wild type protein have been investigated through circular dichroism. GuHCl denaturation experiments monitored by CD showed that cleaved L30 F85W is slightly more stable than cleaved L30 wild type and cleaved L30 F85H. Renaturation experiments showed that denatured cleaved L30 wild type was refolded more efficiently by trimethylamine-N-oxide (TMAO). This suggested that L30 F85W was more stable and in a slightly different conformation. Also, CD scans of L30 mutants and wild type showed secondary structural shifts in the presence of L30 RNA which indicated that the protein changes its conformation upon binding.
To provide supplemental data and an equilibrium alternative to the filter binding experiments, a fluorescence binding assay was developed and implemented. When the RNA binds and brings the nucleotides close to a fluorescently labeled cysteine, the fluorescence of the fluorophore is quenched by the purine-rich RNA internal loop. Through in vitro fluorescence-quenching RNA titrations the relative binding strengths of several mutants were estimated and the relative Kd values were compared to the dissociation constants determined from filter binding data. The binding order from strongest to least binding is L30 wild type, L30 F85H, L30 F85I, L30 F85W and L30 F85A where L30 F85W and F85A exhibit the same quenching. With the exception of L30 F85W, the binding order between methods was the same. This provided support for a fluorescence quenching assay using maleimide dyes tethered to a protein that binds RNA. Determining the strength of the L30 RNA/L30 protein interaction using fluorescence at equilibrium in solution and without the use of radioactive labels could be a safe and efficient method of studying this system and other protein/RNA binding systems.
Selah, Cheryl A. "The Further Characterization of Saccharomyces cerevisiae Ribosomal Protein L30 and Its Interaction with the RPL30 Transcript through Mutational Analysis, Circular Dichroism, and Fluorescence Methods." PhD diss., Bryn Mawr College, 2007.