(59d) Uncovering Structure/Function Design Principles of RNA Transcriptional Attenuators Using Shape-Seq to Characterize RNA Structures in Vivo

RNA gene regulators are emerging as powerful parts for engineering gene regulation and networks. A potential advantage to using RNA regulators over their protein counterparts is the ability to uncover structure/function relationships and infer functional design principles using next generation tools that have recently been developed to characterize RNA structures in vivo (1). A particularly versatile RNA regulator is the pT181 transcriptional attenuator - an RNA sequence in the 5’ untranslated region of a transcript that regulates transcription elongation in response to an antisense RNA expressed in trans. In previous work, a library of orthogonal transcription attenuators was engineered by replacing the antisense-RNA sensing region of the pT181 transcriptional attenuator with the sensing regions from five different naturally occurring antisense-RNA translational regulators (2). The previous study produced a number of regulators that varied in attenuation capability and orthogonality. Some hypotheses were made about how the predicted secondary structures of the attenuators caused the variation, though we had no quantitative method to assess these structure/function relationships. In this work, we chemically probe the structures of these regulators using a newly developed in-cell SHAPE-Seq technique (1) in order to understand the structure/function relationship of these regulators. In-cell SHAPE-Seq provides nucleotide-resolution chemical reactivity spectra for RNAs that reflect their structural state within the cell. By probing the attenuator, antisense, and interacting attenuator-antisense RNA’s of both functional and non-functional regulators, we show that we can directly uncover important structural features required for proper transcription attenuation. This knowledge builds our understanding of how these RNAs function, and brings us closer to a design paradigm that uses experimentally derived structure/function relationships to design chimeric attenuators in silico. Finally, our approach of combining functional experiments with in-cell SHAPE-Seq as a tool for understanding the connection between RNA structure and function is completely general, and represents a quantitative approach to rationally engineering regulatory RNAs.

(1) Watters K. E., et al., “In-cell RNA Structure Probing with SHAPE-Seq,” submitted to the 2014 AIChE National Meeting (TA006), Atlanta, GA (Nov 16-21, 2014).

(2) Takahashi M. K., and J. B. Lucks, “A Modular Strategy for Engineering Orthogonal Chimeric RNA Transcription Regulators,” Nucleic Acids Res, 41 (15) pp. 7577-7588 (Aug. 2013).