Using Analytical Centrifugation to Identify and Study Kink-turned RNA

Posted May 12th, 2010 at 1:34 pm.

Ashton Shaffer
Mentor: Dr. Susan White

RNA (ribonucleic acid) makes it possible for the genetic instructions encoded in DNA to be used to assemble essential molecules like proteins. RNA’s structure allows it to participate in reactions like transcription, which is basically the production of a complementary copy of DNA, and splicing, which is the processing and editing of RNA prior to use for protein production. RNA’s secondary structure, when it is double stranded, tends to consist of nucleotides that base pair in the traditional Watson-Crick manner, where hydrogen bonds are formed only between guanine-cytosine and adenine-uracil, and form a helical configuration. It can, however, have a kink-turn, which contains three unpaired nucleotides followed by several nucleotides that do not exhibit Watson-Crick base pairing. L30, a protein found in yeast, has been found to autoregulate its transcription and splicing by binding to its own RNA transcript, which forms a kink-turn. Learning more about this mechanism of identification, binding, and regulation will provide insight into RNA-protein interactions, which makes kink-turned RNA and L30 particularly interesting.

Kink-turn RNA has previously been studied using radioactive isotopes in gel mobility experiments where the differences are only detected in the presence of Magnesium (Mg2+).
Our objective is to identify RNA with kink-turns without using techniques like radioactive gel electrophoresis and instead utilizing a process called analytical centrifugation. This alternate approach is based on the biophysical principles of hydrodynamics and observes the rate of migration of RNA as it is spun at high speeds in a fluid environment in order to infer its structure. We plan to test it both with and without Mg2+ in solution with the RNA to determine whether or not there are any discernable differences in the resulting RNA mobility. It has the added advantage of permitting us to study RNA in an environment that closely resembles what is actually present in a cell. Our hope is that analytical centrifugation will enable us to not only distinguish the difference between kink-turned and base paired RNA, but also to tell to what degree the RNA is kink-turned (larger and smaller angles). We are also interested to observe whether or not Mg2+ must be present in order to make these distinctions. Being able to differentiate kink-turned and base-paired RNA adds to our general understanding of how structural changes affect the ability of proteins and RNA to interact and the chemical mechanisms of transcription, splicing, and regulation.

Filed under: 2008,Shaffer, Ashton,White, Dr. Susan by Ann Dixon

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