Computational And Experimental Frameworks To Understand The Mechanism Of +1 And -1 Programmed Ribosomal Frameshifting

Abstract

Programmed ribosomal frameshifting (PRF) is an extension of genetic decoding that allows a ribosome to produce inframe products and frameshift products from a single transcript. In +1 PRF, the ribosome moves one nucleotide to the 3’-end of the mRNA while in -1 PRF, the ribosome slips one nucleotide to the 5’-end of the mRNA during translation. Organisms from virus to prokaryotes to human have genes known to involve PRF. This dissertation presents the development of computational and experimental tools to systematically analyze the mechanism of +1 PRF and -1 PRF. The computational tools include: (1) a kinetic model for +1 PRF that reveals the synergistic effects of ribosome E-, P-, and A-sites on promoting +1 frameshift efficiency; (2) a mechanism-based bioinformatic program FSscan that identifies novel +1 frameshift cassettes in yehP, pepP, and cheA genes in Escherichia coli; and (3) a kinetic model for -1 PRF that predicts translation elongation steps significantly affecting -1 frameshift efficiency and the percentage of two types of -1 frameshift products. To confirm model predictions, a dual fluorescence reporter system is developed in E. coli and Saccharomyces cerevisiae. Additonally, -1 frameshift proteins are purified and analyzed by nano-flow liquid chromatography electrospray tandem mass spectrometry to obtain the percentage of the two types of -1 frameshift proteins. Using the reporter system in E. coli, the experimental results are consistent with model predictions. The combination of computational and experimental works accelerates the investigation and expands the range and depth of the understanding. These tools can be further adapted to explore PRF in different organisms or to discover compounds altering PRF efficiency in a high-throughput manner. This work is an example of using systems biology approach to improve our understanding of a complex, but critically important, biological process.