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

RNA plays a central role in gene regulation and information processing in all kingdoms of life. Found in 5\u27 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

Single-Molecule Studies of the Lysine Riboswitch Reveal Effector-Dependent Conformational Dynamics of the Aptamer Domain

The lysine riboswitch is a cis-acting RNA genetic regulatory element found in the leader sequence of bacterial mRNAs coding for proteins related to biosynthesis or transport of lysine. Structural analysis of the lysine-binding aptamer domain of this RNA has revealed that it completely encapsulates the ligand and therefore must undergo a structural opening/closing upon interaction with lysine. In this work, single-molecule fluorescence resonance energy transfer (FRET) methods are used to monitor these ligand-induced structural transitions that are central to lysine riboswitch function. Specifically, a model FRET system has been developed for characterizing the lysine dissociation constant as well as the opening/closing rate constants for the <i>Bacillus subtilis lysC</i> aptamer domain. These techniques permit measurement of the dissociation constant (<i>K</i>_D) for lysine binding of 1.7(5) mM and opening/closing rate constants of 1.4(3) s^–1 and 0.203(7) s^–1, respectively. These rates predict an apparent dissociation constant for lysine binding (<i>K</i>_D,apparent) of 0.25(9) mM at near physiological ionic strength, which differs markedly from previous reports

Modularity of Select Riboswitch Expression Platforms Enables Facile Engineering of Novel Genetic Regulatory Devices

RNA-based biosensors and regulatory devices have received significant attention for their potential in a broad array of synthetic biology applications. One of the primary difficulties in engineering these molecules is the lack of facile methods to link sensory modules, or aptamers, to readout domains. Such efforts typically require extensive screening or selection of sequences that facilitate interdomain communication. Bacteria have evolved a widespread form of gene regulation known as riboswitches that perform this task with sufficient fidelity to control expression of biosynthetic and transport proteins essential for normal cellular homeostasis. In this work, we demonstrate that select riboswitch readout domains, called expression platforms, are modular in that they can host a variety of natural and synthetic aptamers to create novel chimeric RNAs that regulate transcription both <i>in vitro</i> and <i>in vivo</i>. Importantly, this technique does not require selection of device-specific ″communication modules″ required to transmit ligand binding to the regulatory domain, enabling rapid engineering of novel functional RNAs

Deep scanning lysine metabolism in Escherichia coli

Our limited ability to predict genotype-phenotype relationships has called for strategies that allow testing of thousands of hypotheses in parallel. Deep scanning mutagenesis has been successfully implemented to map genotype-phenotype relationships at a single-protein scale, allowing scientists to elucidate properties that are difficult to predict. However, most phenotypes are dictated by several proteins that are interconnected through complex and robust regulatory and metabolic networks. These sophisticated networks hinder our understanding of the phenotype of interest and limit our capabilities to rewire cellular functions. Here, we leveraged CRISPR-EnAbled Trackable genome Engineering to attempt a parallel and high-resolution interrogation of complex networks, deep scanning multiple proteins associated with lysine metabolism i

Codon Compression Algorithms for Saturation Mutagenesis

Saturation mutagenesis is employed in protein engineering and genome-editing efforts to generate libraries that span amino acid design space. Traditionally, this is accomplished by using degenerate/compressed codons such as NNK (N = A/C/G/T, K = G/T), which covers all amino acids and one stop codon. These solutions suffer from two types of redundancy: (a) different codons for the same amino acid lead to bias, and (b) wild type amino acid is included within the library. These redundancies increase library size and downstream screening efforts. Here, we present a dynamic approach to compress codons for any desired list of amino acids, taking into account codon usage. This results in a unique codon collection for every amino acid to be mutated, with the desired redundancy level. Finally, we demonstrate that this approach can be used to design precise oligo libraries amendable to recombineering and CRISPR-based genome editing to obtain a diverse population with high efficiency

Genome-Wide Tuning of Protein Expression Levels to Rapidly Engineer Microbial Traits

The reliable engineering of biological systems requires quantitative mapping of predictable and context-independent expression over a broad range of protein expression levels. However, current techniques for modifying expression levels are cumbersome and are not amenable to high-throughput approaches. Here we present major improvements to current techniques through the design and construction of <i>E. coli</i> genome-wide libraries using synthetic DNA cassettes that can tune expression over a ∼10⁴ range. The cassettes also contain molecular barcodes that are optimized for next-generation sequencing, enabling rapid and quantitative tracking of alleles that have the highest fitness advantage. We show these libraries can be used to determine which genes and expression levels confer greater fitness to <i>E. coli</i> under different growth conditions

Rapid and Efficient One-Step Metabolic Pathway Integration in <i>E. coli</i>

Methods for importing heterologous genes into genetically tractable hosts are among the most desired tools of synthetic biology. Easy plug-and-play construction methods to rapidly test genes and pathways stably in the host genome would expedite synthetic biology and metabolic engineering applications. Here, we describe a CRISPR-based strategy that allows highly efficient, single step integration of large pathways in <i>Escherichia coli</i>. This strategy allows high efficiency integration in a broad range of homology arm sizes and genomic positions, with efficiencies ranging from 70 to 100% in 7 distinct loci. To demonstrate the large size capability, we integrated a 10 kb construct to implement isobutanol production in a single day. The ability to efficiently integrate entire metabolic pathways in a rapid and markerless manner will facilitate testing and engineering of novel pathways using the <i>E. coli</i> genome as a stable testing platform