Characterization of the Binding Affinity of L30e Mutants with KT and BP RNA

Posted June 25th, 2010 at 10:33 am.

Abstract: Ashton Shaffer
Mentor: Dr. White
Ribonucleic acid (RNA) protein interactions are biologically significant due to their role in genetic regulation. In Sacchaomyces cerevisiae (yeast), the ribosomal protein L30 (previously L32) acts as an autoregulator by binding to and splicing its RNA . The binding recognition and affinity depends on the tertiary structures of both the protein (L30e) and RNA (L30). The L30 protein-RNA binding site has been previously studied , revealing a kink-turn motif in the RNA. A kink-turn is characterized by a canonical stem of Watson-Crick paired bases, three unpaired nucleotides, and a purine-rich non-canonical stem that does not conform to Watson-Crick base pairing rules. X-ray crystallography of the L30e protein indicates that the residue in position 85 interacts with the kink-turned region of the RNA (see Figure 1). We intend to study this L30 binding site under various experimental conditions in order to gain further insight into the form and function of this RNA-protein complex.

The first step of our research project is to produce and purify the desired RNAs and proteins. We will use E. coli plasmids that have previously been made in the White Lab Group to make several protein variants of the wild type with mutations of the phenylalanine at the 85th position to alanine, tryptophan, and histidine (WT, F85A, F85W, and F85H respectively). Each of these L30 mutants is attached to maltose-binding protein (MBP) and can be purified by amylose column affinity chromatography. We will transcribe larger (150-200 bp) base paired (BP) and kink-turned (KT) RNA using DNA mutants previously made in the White Lab Group and purify them by filtration using Ambion Megaclear kits. We will also use oligonucleotide primers to synthesize small RNAs (~50 bp). The second step of this summer’s project is to test the binding affinity of each of these protein mutants for the BP and KT RNA using a variety of methods. We plan to use Isothermal Calorimetry (ITC) to measure the enthalpy (H) of the protein-RNA binding complex. We will also conduct bandshift assays with radiolabeled RNA and the L30-MBP to qualitatively compare the binding affinities of the different protein mutants for the RNAs. Similarly, we intend to utilize column chromatography to quantitatively compare binding of fluorescently tagged L30-MBP protein with its RNA.

Filed under: 2010,Shaffer, Ashton,White, Dr. Susan Tags: by Lisa Klinman

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