Poster abstracts

Poster number 15 submitted by David Bushhouse

Expression platform features tune functional characteristics of the Clostridium beijerincki ZTP riboswitch

David Z. Bushhouse (Interdisciplinary Biological Sciences Graduate Program, Northwestern University; Center for Synthetic Biology, Northwestern University), Julius B. Lucks (Center for Synthetic Biology, Northwestern University; Department of Chemical and Biological Engineering, Northwestern University)

Abstract:
Riboswitches are short cis-encoded RNA elements that regulate the expression of downstream genes in response to small molecule ligands.1 While much is known about the structural nature of riboswitch-ligand interactions, we are only beginning to understand how the dynamic process of co-transcriptional RNA folding leads to regulatory outcomes.2 To elucidate principles of expression platform function, we are investigating the Clostridium beijerincki ZTP riboswitch, a transcriptional ON switch that senses ZTP (~115μM EC50) as an indicator of folate stress.3 In the absence of ZTP/ZMP, the formation of a strong intrinsic terminator attenuates transcription; however, in the presence of ZTP/ZMP, the formation of this hairpin is delayed by a ligand-dependent alternative folding pathway, allowing RNA polymerase to process past the termination site.4 Previous co-transcriptional structure probing studies implicated strand displacement as the primary gene regulatory mechanism. How the strand displacement conformational change is controlled by the expression platform, however, remains poorly understood.4 Using an in vivo reporter assay and a functional mutagenesis approach, we have found that the length of the terminator hairpin loop can widely tune riboswitch sensitivity (~30–220 μM EC50), extensions to the terminator hairpin affect dynamic range (~3–15-fold), and strategically placed mismatches and bulges within the terminator stem can widely alter baseline expression and dynamic range (~2–19-fold). Together, our findings show that the Cbe ZTP expression platform tunes the functional characteristics of the riboswitch via a concerted combination of sequence and structural elements to achieve the baseline expression, sensitivity, and maximal response amenable to the organism. These results also indicate possible approaches for the optimization of natural riboswitches that employ strand displacement mechanisms for biotechnology applications.

References:
1. Breaker, R. R. Riboswitches and the RNA world. Cold Spring Harbor Perspectives in Biology 4, 1–15 (2012).
2. Watters, K. E., Strobel, E. J., Yu, A. M., Lis, J. T. & Lucks, J. B. Cotranscriptional folding of a riboswitch at nucleotide resolution. Nature Structural and Molecular Biology 23, 1124–1131 (2016).
3. Kim, P. B., Nelson, J. W. & Breaker, R. R. An ancient riboswitch class in bacteria regulates purine biosynthesis and one-carbon metabolism. Molecular Cell 57, 317–328 (2015).
4. Strobel, E. J., Cheng, L., Berman, K. E., Carlson, P. D. & Lucks, J. B. A ligand-gated strand displacement mechanism for ZTP riboswitch transcription control. Nature Chemical Biology 15, 1067–1076 (2019).

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