Abstract: Natalia Mavrogiannis
Mentor: Dr. White
Ribonucleic Acid (RNA) is a biologically important macromolecule, which codes for the protein production in organisms. RNA, much like proteins, forms a tertiary structure. One such tertiary conformation of RNA is a kink-turn (KT). This KT region of the RNA consists of Watson-Crick base pairs that surround a bulge consisting of non-base paired nucleotides in the RNA, this bulge results in a sharp bend in the phosphodiester backbone. This region is of interest to our research project because proteins bind to RNA at the KT. The proteins that interact with KT RNA are structured to recognize this unique shape and interact with the RNA to form RNA-protein complexes. The protein L30e, an autoregulatory ribosomal protein, is known to bind to its RNA. This RNA-protein interaction then inhibits splicing of the transcript RNA and the binding of the protein to its mRNA represses translation. Previous experiments have characterized this interaction using Saccharomyces cerevisiae ribosomal protein L30 (previously L32) .
Our goal is to study the RNA-protein complex formed by the protein L30e and its mRNA using various mutated forms of the protein that we will produce and purify using previously designed L30e plasmids. We intend to study RNA-protein complex using the wild-type L30e protein (shown below) as well as three proteins mutated at position 85 from phenylalanine to alanine (F85A), histidine (F85H), and tryptophan (F85W) to find if these mutations affect binding. The first task is to produce the wild type and mutant protein and then subsequently purify it. We used the designed plasmids and over-expressed the L30 protein. We then employed the use of an Amylose Affinity column to purify the protein because each L30 protein is attatched to Maltose Binding Protein (MBP). We then intend to study the RNA-protein complex binding using radioisotopes and fluorescence experiments. We will use radiolabeled RNA to determine the strength of the interaction between the RNA and the protein by obtaining band shift assays. We will use fluorescence experiments to tag the protein, which will also allow us to track the strength of the RNA protein interaction. We will also employ the techniques of sedimentation, which allows us to determine of the shape of the RNA as it settles in solution, and Isothermal Calorimetry (ITC) to study the biophysical properties of the RNA-protein binding.
Image of the L30 protein, in green (phenylalanine highlighted in yellow) bound to kink-turned RNA (shown as a multi-colored molecule).