Discipline: Biological Sciences
Claudia Alvarado - University of Georgia
Co-Author(s): Cory Momany and Nick Galloway, University of Georgia, Athens, GA
RNA plays a central role in many biological processes with their ability to be genetic information to catalyze reaction as ribozymes and regulate expression of macromolecules. The number of non-coding RNAs (ncRNA) is currently unknown and only a few ncRNA 3D structures have been developed. A potentially viable approach to addressing this issue is through structure prediction with computer modeling. Such predictions may help narrow structure candidates for further experimental validation. Current state of the art methods have yet to deliver predictions for RNAs of lengths beyond 50. The goal of our prediction methods will consider sequence information to be combined with biological and mathematical insights to provide structural prediction of ncRNA. RNA Sequences of varying lengths and various strictures, including hairpin, junctions, and pesudoknots of highly conserved bacterial RNA families will be explored. The structures will not only serve to analyze computational results but they will also be coerced to new motifs and nucleotide interactions. Structures proposed will be validated using RNA experimentation biological properties of ncRNAs. Solved 3D structures will provide insight into interplay between sequences and structural conservation during ncRNA evolution. Rendering integration of RNA bioinformatics model predictions with X-ray phasing as an approach to predict protein structures and molecular placement. The project goals include expressing, crystalizing and determining structures for E.coli sRNA families: Spot-42, GcvB, and RyhB. RNA preparation via in vitro transcription using T7 RNA polymerase, followed by purification on denaturing polyacrylamide gel electrophoresis (PAGE). Other methods to be completed include 1) use of in vivo expression of RNA molecules engineered inside tRNA anticodon loops; 2) co-transcription of up- and down- stream ribozymes or selective RNAses; and 3) a variety of crystallization chaperones. Approaches of mass RNA production will be reproduced and implemented for propagation and purification procedure will be done using Golden Gate cloning. In vitro production will require appropriate dNA segments to encode RNA molecules in t7 promoters/terminators and ribozymes to be creates using PLC amplification. In vivo expression, calls for T7 components to be duplicated I tandem into a vector. RNA will be produced in transformed RNAse E deficient E.coli strain will be optimized for high nucleic acid production. Complementary DNA sequence constructs will anneal to RNA molecules to allow for further DNA/RNA hybrids to be created. Quality of all samples is to be characterized by PAGE, CD spectroscopy and dynamic light scattering. RNA will be used in initial phases of crystal structure determination and for computational comparison purposes.
Funder Acknowledgement(s): National Science Foundation / University of Georgia, College of Biomedical and Pharmaceutical Sciences
Faculty Advisor: Cory Momany,