Using Isothermal Calorimetry to Study the Thermodynamics of RNA-Protein Binding

Posted June 24th, 2010 at 12:40 pm.

Abstract: Alexandra Raeber
Mentor: Dr. White
Interactions between ribonucleic acid (RNA) and protein, which are dependent upon the three-dimensional structures of these molecules, are an important component of cellular activity. L30e, a yeast ribosomal protein, autoregulates its production by binding to its RNA transcript. While most double-stranded RNA forms traditional Watson-Crick base pairs, some RNAs, like L30e, have a kink-turn, a region of non-Watson-Crick base pairs and non-base paired nucleotides that create a sharp bend in the RNA. The L30e protein binds to kink-turn in L30e RNA to form a complex. In order to study this binding several types of L30e protein and L30e RNA will be produced. Mutant and wild-type L30e protein will be produced by inducing the over expression of the protein in a bacterium bearing a plasmid which codes for it. The protein will by purified using affinity chromatography. L30e RNA will be produced by digesting and then transcribing the same type of plasmid DNA that is used to produce the protein. The RNA will be purified using a MEGAclear purification kit.

The goal of this summer’s research is to gain a better understanding of the thermodynamics of L30e RNA-protein interaction by measuring ΔH and ΔS of binding using isothermal titration calorimetry (ITC), and from there, determining ΔG of binding. This will hopefully provide useful biophysical information about L30e RNA. ITC is a technique in which a one molecule is added in known increments to a constant quantity of another molecule to which it is capable of binding and the amount of heat that must be added or removed in order to keep the temperature of the reaction stable is measured. In order to gain proficiency in ITC, test reactions using sucrose, EDTA or EGTA, and RNase pCp will be run. Next, the data from these tests will be used to learn how to find ΔH and ΔS from ITC data. After doing these test reactions, it will hopefully possible to get useful data from RNA-protein binding experiments. These experiments will involve several mutants of L30e RNA and several mutants of L30e protein that were made earlier in the summer. The binding between different RNA mutants and magnesium ions (Mg2+) will also be tested. In doing these experiments we hope to gain a better understanding of how the thermodynamics of RNA-protein binding is affected by mutations in the primary structure of the protein and the sequence of the RNA to which it is bound, as well as how RNA and Mg2+ interact.

Filed under: 2010,Raeber, Alexandra,White, Dr. Susan Tags: by Lisa Klinman

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