A Thermal Denaturation method for distinguishing between K-turn and non-K-turn RNA

Posted May 14th, 2010 at 10:43 am.

Amber R. Moore

Mentor: Dr. Susan A. White

clip_image002_000 Abstract. Protein-RNA interactions are important for cellular growth and regulation. Proper interactions between protein and RNA require both of their interfacial sites to be conserved for specific recognition and binding. Organisms, like Saccharomyces cerevisiae (yeast), have evolved in ways that condense multi-component and multi-step pathways into more self-contained processes. This allows the cell machinery to maintain, if not improve, its specificity and regulation, while increasing the efficiency of these vital mechanisms. Knowledge of protein and RNA interactions continues to aid in the development of therapeutic approaches for the treatment of various diseases.

clip_image005The S. cerevisiae autoregulatory ribosomal protein L30e, or RPL30, is capable of inhibiting the splicing and translation of its own transcript and mRNA by forming a protein-RNA complex. The L30e protein has a single domain composed of four beta sheets and four alpha helices. The L30e RNA is present in two strands and assumes a helix-internal loop-helix motif, commonly referred to as the kink-turn, or K-turn motif. The internal loop, comprised of one strand with unpaired nucleotides and a second strand with missing nucleotides, connects a canonical stem (Watson-Crick base paired helix) to a non-canonical stem (helix containing some non-Watson-Crick base pairs). L30e recognizes and binds to the K-turn RNA when there is an excess of L30e protein (not being incorporated into the ribosome). This forms the L30e protein-RNA complex that represses further protein expression. We expect to find that any RNA sequence mutation altering the K-turn will disturb the protein-RNA interaction of the L30e complex.

Thermal denaturation will be the applied method in differentiating between K-turn RNA and non-K-turn RNA while Mg2+ is present to simulate cellular conditions and assist the K-turn RNA in maintaining its sharp bent structure. Results are expected to show differences in the stabilities among the RNA variants being tested: BP, KT12, and KTAU. The BP (base paired) variant has complete Watson-Crick base pairing among the nucleotides, whereas the KT (kink turn) variant has non-Watson-Crick base pairing which causes the helix-internal loop-helix motif in the RNA. KT12 has an adenine (A) replaced with a uracil (U) at the twelfth nucleotide position in the K-turn region of the RNA. KTAU has the same A to U mutation, but it occurs outside of the K-turn region. Thermal denaturation is not only expected to show differences in stability between the BP and KT RNA, but also between the KT variants with nucleotide mutations inside and outside of the K-turn region.

Filed under: 2008,Moore, Amber,White, Dr. Susan by Ann Dixon

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