Project Description
The purpose of this project is to understand why organisms modify the ends of their RNA molecules. Until recently, most scientists believed that cells only modify the RNA 5’-end using a 7-methly GDP to cap RNA molecules that are used as templates for protein synthesis on ribosomes. In the past decade, however, other 5’end modifications have been found including nicotinamide adenine dinucleotide (NADH), which is known mainly as a valuable co-enzyme used to transform sugar into energy. Our lab discovered the enzyme that removes NADH caps from RNA, which is a Nudix enzyme called NudC prokaryotes. We suspect that a similar protein called Nudt12 performs the same function in humans. The goal of this project is to understand how NudC & Nud12 identify rare NADH-capped RNA in the ocean of other cellular RNA molecules. The proteins share two motifs than might be important, one in the Nudix domain, and a second in a “zinc-finger” domain. Our plan is to use site-directed mutagenesis to alter the DNA plasmids encoding NudC and Nudt12 so they no longer contain one of the two possible RNA-binding motifs, purify the proteins, and test their ability to bind zinc, NADH-capped RNA and remove the caps.
Tasks and Responsibilites
The student will learn DNA cloning, PCR, recombinant DNA technology, protein expression, protein purification, and a variety of enzyme assays. Specifically, they will design oligonucleotides to introduce mutations, perform PCR, digest PCR products with restriction enzymes to remove the wildtype DNA, and transform E. coli with the products. They will then use DNA sequencing to identified clones of interest, transform an expression strain with the proper plasmid, and use it to express proteins in large cultures. E. coli expressing the mutant proteins will be lysed, and the proteins purified using affinity, ion exchange and gel filtration chromatography. DNA binding will be monitored using gel-shift and fluorescence polarization assays, and data analyzed using non-linear regression analysis to determine the dissociation constant and free energy of protein-metal and protein-nucleic acid interactions.
Desired Qualifications
None listed.