Engineering the PP7 Phage Coat Protein for Imaging of MRNAs in living cells

Chao, Jeffrey

Abstract

Fluorescence microscopy is a powerful tool for quantitatively measuring biological processes in their cellular context as they take place. The bacteriophage MS2 coat protein when fused to a fluorescent protein allows for the detection of RNAs containing its cognate RNA binding site. This technique allows for the fate of individual mRNAs to be followed in real time in living cells and has made possible the interrogation of gene expression at the single cell and single molecule level. In order to extend this methodology and take advantage of the spectral array of fluorescent proteins, the initial aim of this proposal is to engineer the bacteriophage PP7 coat protein and its translational operator for live cell imaging of RNA. The ability for multicolor labeling of mRNAs will allow more sophisticated experiments characterizing gene expression to be performed. Reporter constructs will be site-specifically inserted into the yeast genome to measure the transcriptional activity of individual alleles for genes with differing function. A single mRNA will be labeled in two positions, an intron and exon, to follow the splicing reaction in mammalian cells. This technology may also have uses in yeast three-hybrid, affinity chromatography and tethering experiments in which the MS2 system has been utilized. Unlike the MS2 coat protein interaction with its RNA target, much less is known about the molecular interactions that determine the PP7 coat protein's specificity for its RNA stem-loop. Structural and biochemical studies of the complex will allow the specificity determinants to be elucidated. This work may allow the rational design of novel RNA-protein complexes for use in live cell imaging of RNA. ? ? Understanding the mechanism of gene expression will facilitate the therapeutic use of the information provided by genome sequencing. The primary goal of this proposal is to generate reagents that allow gene expression to be quantitatively characterized in real-time in living cells. ? ? ?