Sequence Specificity in RNA-Mediated Transcriptional Attenuation Examined by Coarse-Grained MD Simulations

Abstract

In RNA-mediated transcriptional attenuation, the elongation of a nascent mRNA is halted prematurely through the binding of a non-coding, antisense RNA. This process is initiated by a kissing loop complex between the antisense and target hairpins. Complementarity then favors extended inter-strand base pairing; however, transcriptional attenuation requires rapid kinetics, which depend highly on sequence. Computational prediction of sequences that rapidly form extended complexes would aid in the rational design of mutually orthogonal regulators, allowing their simultaneous application to synthetic systems without generating crosstalk. Additionally, probing the energetics of these systems provides insight into the fundamental relationship between RNA sequence, structure and function.Here, we employ coarse-grained molecular dynamics (MD) simulations to investigate the potential of synthetic RNA hairpins, derived from prokaryotic attenuation systems such as pT181, to form extended complexes with their antisense partners. The resulting trajectories of an experimentally known RNA-attenuator system show a strong propensity for extended inter-strand base pairing and the formation of stable intermediates. In contrast, a sequentially similar, but non-functional system shows almost no potential for extended inter-strand base pairing. This observation was consistent even when the system was subjected to increased temperatures and external pulling forces that greatly increased the rate of complex formation in the known attenuator system. These results suggest that changes in the energetic landscape of inter-strand base pairing can be captured by coarse-grained MD simulations and provide a route for the prediction of new, orthogonal sequences capable of transcriptional attenuation in synthetic systems.