Mentor: Dr. Susan White
Ribonucleic acid (RNA) allows for the production of proteins, which aid in the cellular growth and regulation, through the utilization of the genetic coding present in DNA during transcription and the processing and editing of the RNA structure during splicing. The double stranded structure of the RNA customarily consists of the Watson-Crick base-pairing of the purine and pyrimidine nucleotides—hydrogen bonds between adenine-uracil, and guanine- cytosine. However, the nucleotides may not pair traditionally; one example among many is helix-internal loop-helix motif (the kink-turn motif). The internal loop consists of a strand of unpaired nucleotides, which follows a Watson-Crick base-paired stem and precedes a non-Watson Crick base-paired stem. Advances in the understanding of the structure and binding affinities of the K-turn in comparison to those of the Watson-Crick representative RNA (base-paired RNA) enhance the understanding of the RNA-protein interactions.
To test mobility of the kink-turn and base-paired RNA, we will utilize Analytical Ultracentrifugation and Gel Filtration Chromatography. Using Analytical Ultracentrifugation, sedimentation experiments can extract the rate of migration and possibly be used in the determination of shape as the RNA settles in solution. The rate of sedimentation is dependent on the shape and density of the molecule, as each differently sized molecules settle at different minimal centrifugal forces—dense, spherical molecules sediment at a more rapid rate, while elongated molecules at a slower rate. Our hope is to determine whether kink-turn and base-paired RNAs sediment differently in the presence of Mg2+. We will test the Analytical Ultracentrifugation with Mg2+ in solution with the kink-turn and base-paired RNA to determine any difference in mobility of the two forms of RNA. When binding to Mg2+, kink-turn RNA is assumed to be bent and “folded over,” suggesting a more rapid sedimentation than the base-paired RNA, which is more elongated. The gel filtration chromatography allows for analysis of mobility in the two RNA forms through chromatography columns. Greater mobility depends on the more condensed shape of the molecule traveling through the column. Our hope is to determine whether the kink-turn and base-paired RNAs have different mobilities through the columns. If there is a difference, is magnesium and heat required to maintain that difference? Testing this approach with both forms of RNA with and without the presence of magnesium at two main temperatures, we predict that the K-turn RNA, having a “folded over structure” will have a greater mobility than the base-paired RNA. Both the analytical centrifugation and the gel filtration chromatography help to determine the size relationship between the two forms of the RNA, and indirectly aid in the determination of structure and binding capabilities.