Mechanistic Insights into Molecular Recognition and the Regulatory Landscape of the lysine Riboswitch

Garst, Andrew D.

Graduate Thesis Or Dissertation

Mechanistic Insights into Molecular Recognition and the Regulatory Landscape of the lysine Riboswitch Public Deposited

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Citeable URL: https://scholar.colorado.edu/concern/graduate_thesis_or_dissertations/6395w7238

Abstract

RNA plays a central role in gene regulation and information processing in all kingdoms of life. Found in 5' leader sequence of many bacterial mRNAs, riboswitches serve as an exemplar of this duality as they control expression of their own transcript by directly binding small molecule metabolites in the cell. These RNAs adopt tertiary structures to scaffold highly specific ligand binding sites, reminiscent of their protein counterparts. Ligand binding is then coupled to conformational changes in the RNA that influence expression of the downstream message by altering the transcription or translation of the message. To investigate the structural basis by which bacterial mRNAs specifically recognize lysine the aptamer domain from the leader sequence of the asd gene in Thermatoga maritima was solved by X-ray crystallography in both the free and bound conformations. These structures were complemented with solution based approaches to demonstrate that the tertiary architecture of the lysine aptamer domain is largely preorganized at 5 mM Mg2+ in the absence of ligand. Ligand binding was found to induce limited conformational changes within the five-way junction of the RNA. Based on these collective observations a site-specifically labeled RNA construct was designed to enable further thermodynamic and kinetic analysis of ligand binding to the aptamer. Using a series of lysine analogs that challenge key aspects of the structural model, we obtained a detailed understanding of the energetics of ligand recognition and demonstrate the importance of solvent and ion-mediated contacts in achieving a high affinity interaction. The binding kinetics of these analogs were also used to develop a simple mathematical framework for predicting the regulatory behavior of the RNA during transcription. Kinetic predictions were tested using a minimally reconstituted in vitro transcription assay to gain further empirical insight into the regulatory functions of the B. subtilis lysine riboswitch and correlate the biological function with studies of the isolated aptamer. The regulatory response of lysine was found to agree well with a simple two state mechanism of ligand binding for lysine at a variety of NTP concentrations. The five fold variation in T50 observed for lysine along with changes in the observed termination efficiency also suggest that this RNA by indirect means integrates a more global picture of metabolism into its regulatory response. Kinetic predictions were also predictive for the regulatory response of many of the alternative ligands at low NTP concentrations, but were found to be less accurate in predicting responses at elevated NTP concentrations, suggesting that the simple model may neglect certain features of the transcription process. The in vitro transcription assays were also employed to study the mechanism by which ligand binding is coupled to the secondary structural switch in the expression platform. A systematic survey of mutations to the P1 helix demonstrated that this element serves as the primary module for interdomain communication in the natural riboswitches, an insight that facilitated approaches to rationally design and optimize chimeric riboswitches. These studies have collectively shown that the regulatory switch is self contained in the expression platform and can be reprogrammed to be responsive to a large number of alternative ligands through a simple mix and match approach.

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