Talk abstracts
Talk on Saturday 12:00-12:15pm submitted by Kiel Kreuzer
Investigating TRAP-mediated attenuation of the trp operon with cotranscriptional SHAPE-Seq
Kiel D. Kreuzer (Department of Biological Sciences, University at Buffalo), Molly E. Evans (Department of Chemical and Biological Engineering, Northwestern University), Julius B. Lucks (Department of Chemical and Biological Engineering, Northwestern University), Paul Gollnick (Department of Biological Sciences, University at Buffalo)
Abstract:
In Bacillus subtilis, the trp RNA-binding attenuation protein (TRAP) regulates the expression of the trp (trpEDCFBA) operon that is required for tryptophan biosynthesis1. In excess tryptophan, the 11-mer TRAP is activated to bind to the 11 (G/U)AG triplet repeats in the trp operon leader region and promotes transcription attenuation. The proposed model for this regulatory mechanism involves mutually exclusive RNA structures, where TRAP binding to the leader RNA causes formation of an intrinsic transcription terminator. In the absence of TRAP binding, the terminator is prevented from forming due to overlap with an upstream antiterminator structure that is more thermodynamically stable. Recently, additional studies of TRAP-dependent transcription regulation in vivo and in vitro suggest that the published model of mutually exclusive RNA structures may not fully explain the regulatory mechanism. To further investigate the role of TRAP in RNA structural rearrangement and attenuation during the timescale of transcription, we used cotranscriptional SHAPE-Seq2. This method determines the SHAPE reactivity of nascent RNAs at nucleotide resolution and can reveal folding pathways and kinetically trapped structural intermediates that form as the RNA is transcribed. These structures provide additional information of TRAP-dependent regulation and may be more biologically relevant compared to the structural studies of the RNA under equilibrium conditions. Here, we describe the cotranscriptionally folded trp leader RNA in the presence and absence of TRAP, identify a signature of SHAPE reactivity for TRAP binding, and challenge the existing regulatory model for the role of TRAP in transcription attenuation.
References:
1. Gollnick P. 1994. Regulation of the Bacillus subtilis trp operon by an RNA‐binding protein. Molecular Microbiology. 11: 991-997.
2. Strobel EJ, Watters KE, Nedialkov Y, Artsimovitch I, Lucks JB. 2017. Distributed biotin–streptavidin transcription roadblocks for mapping cotranscriptional RNA folding. Nucleic Acids Res. 45:e109–e109.
Keywords: SHAPE, Termination, Gene regulation