Mentor: Dr. Susan White
X-ray and NMR Refined structure of Yeast L-30e-mRNA Complex. (Chao, 2004) 1
RNA, like proteins, folds into 3D structures, consisting of motifs such as hairpins, pseudoknots, and internal loops. This research project focuses on visualizing one such motif, which is called the kink-turn. The kink-turn region consists of Watson-Crick base pairs on either end of a short asymmetric bulge that consists of non-base paired nucleotides, which results in a sharp bend in the phosphodiester backbone. This region is of particular interest because it has been found that proteins bind to RNA selectively at this region. The figure above shows the yeast L30e protein binding to the kink-turn region of its m-RNA. The biophysical structure characterization of the kink-turn of RNA would thus give information about the nature of this motif and its importance in protein-RNA interactions. Such research will aid in studying potentially useful protein-RNA interactions as well as construction of RNA molecular units capable of self-assembling into RNA nanostructures. The goal of this research is to use Atomic Force Microscopy (AFM) to visualize and characterize RNA molecules that have a kink-turn.
For this purpose, previous work done in the lab involved the construction of a relatively large molecule of double stranded RNA with a kink-turn. Two different sequences of 9 and 6 nucleotides respectively were inserted into commercially provided plasmid using site-directed mutagenesis. Linearization of DNA containing these sequences and subsequent transcription with T7 RNA polymerase resulted in the production of RNA that was approximately 900 nucleotides in length. The region of insertion, because it contained non-complementary sequences, resulted in a kink-turn in the RNA molecule. This molecule is large enough to be visualized with the AFM and to study the degree of bending caused by the kink-turn accurately.
Currently, we are focusing on exploring the technique of atomic force microscopy and using it to visualize DNA samples in order to arrive at a method of sample preparation that is suitable for our goals. The most common way of preparing samples is to use functionalized mica surfaces on which the molecules of interest have been immobilized. This general method has to be made specific to our RNA molecules. Further experiments will involve the visualization of kink-turn and non kink-turn RNA molecules in dry as well as liquid environments, and studying the effect of protein (L30e-Maltose Binding Protein) and metal-ions (Mg2+) on the degree of bending of the kink-turn. It is expected that the protein will bind to the kink-turn region and that this will be visible in the AFM images. It is also anticipated that this will change the degree of bending of the kink-turn and we hope to be able to make accurate measurements of the angle of bending.
1 Chao, J A, Williamson, J R. Joint X-Ray and NMR Refinement of Yeast L-30e-mRNA Complex. Structure. (2004). 12(7): 1165-1176