Poster abstracts
Poster number 93 submitted by Sarah Starcovic
Screening of Group IIC Intron IEP Tags for RNP Kinetic Studies
Sarah A. Starcovic (Department of Biochemistry, West Virginia University), Olivia P. Carpenter (Department of Biochemistry, West Virginia University), Aaron R. Robart (Department of Biochemistry, West Virginia University)
Abstract:
Group II introns are self-splicing ribozymes that catalyze intron self-splicing reactions. Splicing reactions occur through two competing reactions, a non-physiological hydrolytic reaction, and a branched splicing reaction that results in the formation of intron lariats. Group II introns often encode an intron-encoded protein (IEP) which assists splicing. A primitive sub-type of these genetic elements, known as IIC introns, require the IEP to efficiently form intron lariat products. Interestingly, IIC lack many RNA secondary structural features associated with ribozyme catalyzed lariat formation, suggesting that the IEP compensates for the “missing” RNA structure. Due to this ribonucleoprotein (RNP) dependence, IIC introns offer a unique opportunity to understand how self-splicing ribozymes transitioned into the RNP dependent splicing machines found in eukaryotic cells. Here, we report the expression and purification of several different affinity tags attached to the IEP. Our immediate goals are to use these affinity tags to tether the IEP to surface plasmon resonance (SPR) chips to study binding and dissociation kinetics. Each tagged IEP was purified, checked for splicing activity, and if applicable, an RNP pulldown was performed. Use of multiple purification tags will also aid our ongoing RNP purification efforts for cryo-EM structural studies.
Keywords: ribonucleoprotein, SPR, IEP