Poster abstracts
Poster number 121 submitted by Beulah Mae Ann Solivio
Mutagenesis of RNase U2 for Enhanced Specificity
Beulah Mae Ann Solivio ( University of Cincinnati), Balasubramanyam Addepalli ( University of Cincinnati), Patrick Limbach ( University of Cincinnati)
Abstract:
Given several ribonucleases (RNases) that have specific cleavage on Uridine (U), Cytosine (C), and Guanosine (G), we aim to generate a RNase that has specificity to cleave at Adenosine (A). Due to the availability of RNase U2, which cleaves at both A and G with a preference on A, our goal is to modify the nucleotide binding domain of RNase U2 to be selective for A by rational mutagenesis. In order to achieve this goal, we designed molecular models indicating hydrogen-bonding interactions of RNase U2 complexed with 3’-AMP to serve as a guide for mutagenesis. Mutagenesis has been performed with the use of synthetic gene blocks that were inserted into Pet22b plasmids. Further, recombinant plasmids have been transformed onto E. coli, from which the mutant protein have been overexpressed, extracted, and purified. Specificity was determined by digestion of tRNAPhe and analysis with mass spectrometry.
References:
Czaja, R., Struhalla, M., Hoschler, K., Saenger, W., Strater, N., Hahn, U., (2004)
Matsuzaki, T., Saski, C., Uchida, T., Satow, Y., and Noguchi. S., (1995)
Keywords: RNase U2 , mutagenesis, specificity