Post-transcription Initiation Control of Gene Expression

Project Summary/Abstract A unifying theme of this research is to determine how RNA structure and RNA-binding proteins regulate gene expression by affecting the activity of RNA polymerase (RNAP) or by affecting ribosome access to the mRNA. Critical mechanistic insight into these processes will be obtained through studies in Escherichia coli and Bacillus subtilis. We previously developed a transcriptomics 3' end-mapping method called Term-seq to identify >1,500 intrinsic terminators in Bacillus subtilis. Many of these terminators are in 5' leader regions, some of which have been shown to regulate gene expression by attenuation/antitermination mechanisms. Another transcriptomics method that was developed by Philip Bevilacqua and Sarah Assmann at Penn State called Structure-seq is used to probe the structure of RNA in vivo. Structure-seq will be used to develop a method for protein-RNA footprinting in vivo, while both Structure-seq and Term-seq will be used for the discovery of novel RNA-based gene regulatory mechanisms. CsrA is a conserved RNA-binding protein that in E. coli binds to and regulates hundreds of mRNAs by affecting their translation and decay. Since CsrA binds to numerous RNA targets in E. coli many of which have been verified in vitro, we will use Structure-seq to develop a method for genome-wide protein-RNA footprinting in E. coli using CsrA-RNA interaction as a testbed. These footprinting studies will then be extended to a conserved RNA helicase called DeaD. Of particular importance, the footprinting methods will be applicable to essentially any organism or cell type. Another discovery-based theme of this research will combine Structure-seq with Term-seq to identify new RNA-based gene regulatory mechanisms at an unprecedented scale. We will identify regulatory mechanisms that respond to metabolites and stresses such as high temperature, and then determine the underlying molecular mechanisms of regulation. This supplement will provide Dr. Gulshan Ara the opportunity to re-enter into a productive career path towards scientific independence. The experimental plan will augment her existing experience in RNA biochemistry and also extend her expertise into microbiology, transcriptomics, bioinformatics, and RNA-based gene regulatory studies.