Synthetic riboswitches that induce gene expression in diverse bacterial species.

We developed a series of ligand-inducible riboswitches that control gene expression in diverse species of Gram-negative and Gram-positive bacteria, including human pathogens that have few or no previously reported inducible expression systems. We anticipate that these riboswitches will be useful tools for genetic studies in a wide range of bacteria.

A high-throughput screen for synthetic riboswitches reveals mechanistic insights into their function.

Riboswitches are RNA-based genetic control elements that regulate gene expression in a ligand-dependent fashion without the need for proteins. The ability to create synthetic riboswitches that control gene expression in response to any desired small-molecule ligand will enable the development of sensitive genetic screens that can detect the presence of small molecules, as well as designer genetic control elements to conditionally modulate cellular behavior. Herein, we present an automated high-throughput screening method that identifies synthetic riboswitches that display extremely low background levels of gene expression in the absence of the desired ligand and robust increases in expression in its presence. Mechanistic studies reveal how these riboswitches function and suggest design principles for creating new synthetic riboswitches. We anticipate that the screening method and design principles will be generally useful for creating functional synthetic riboswitches.

Genetic screens and selections for small molecules based on a synthetic riboswitch that activates protein translation.

Genetic selection provides the most powerful method to assay large libraries of biomolecules for function. However, harnessing the power of genetic selection for the detection of specific, nonendogenous small-molecule targets in vivo remains a significant challenge. The ability to genetically select for small molecules would provide a reaction-independent mechanism to clone biosynthesis genes from large DNA libraries and greatly facilitate the exploration of large libraries of mutant enzymes for improved synthetic capabilities including altered substrate specificities and enhanced regio- or stereoselectivities. While remarkable progress has been made in developing genetic methods to detect small molecules in vivo, many of these methods rely on engineering small-molecule-protein interactions which remains a difficult problem, and the potential for some of these systems to assay large libraries is limited by the low transformation efficiency and long doubling time of yeast relative to bacteria. Herein, we demonstrate that synthetic riboswitches that activate protein translation in response to a specific small molecule can be used to perform sensitive genetic screens and selections for the presence of small molecules in Escherichia coli. We further demonstrate that the exquisite molecular discrimination properties of aptamers selected in vitro translate directly into an in vivo genetic selection system. Finally, we demonstrate that a cell harboring a synthetic riboswitch with a particular ligand specificity can be selectively amplified from a million-fold larger pool of cells containing mutant riboswitches that respond to a closely related ligand, suggesting that it is possible to use genetic selection in E. coli to discover synthetic riboswitches with new ligand specificities from libraries of mutant riboswitches.

Synthesis and bacterial expression of a gene encoding the heme domain of assimilatory nitrate reductase.

Assimilatory NADH:nitrate reductase (EC 1.6.6.1), a complex Mo-pterin-, cytochrome b(557)-, and FAD-containing protein, catalyzes the regulated and rate-limiting step in the utilization of inorganic nitrogen by higher plants. A codon-optimized gene has been synthesized for expression of the central cytochrome b(557)-containing fragment, corresponding to residues A542-E658, of spinach assimilatory nitrate reductase. While expression of the full-length synthetic gene in Escherichia coli did not result in significant heme domain production, expression of a Y647* truncated form resulted in substantial heme domain production as evidenced by the generation of "pink" cells. The histidine-tagged heme domain was purified to homogeneity using a combination of NTA-agarose and size-exclusion FPLC, resulting in a single protein band following SDS-PAGE analysis with a molecular mass of approximately 13 kDa. MALDI-TOF mass spectrometry yielded an m/z ratio of 12,435 and confirmed the presence of the heme prosthetic group (m/z=622) while cofactor analysis indicated a 1:1 heme to protein stoichiometry. The oxidized heme domain exhibited spectroscopic properties typical of a b-type cytochrome with a visible Soret maximum at 413 nm together with epr g-values of 2.98, 2.26, and 1.49, consistent with low-spin bis-histidyl coordination. Oxidation-reduction titrations of the heme domain indicated a standard midpoint potential (E(o)') of -118 mV. The isolated heme domain formed a 1:1 complex with cytochrome c with a K(A) of 7 microM (micro=0.007) and reconstituted NADH:cytochrome c reductase activity in the presence of a recombinant form of the spinach nitrate reductase flavin domain, yielding a k(cat) of 1.4 s(-1) and a K(m app) for cytochrome c of 9 microM. These results indicate the efficient expression of a recombinant form of the heme domain of spinach nitrate reductase that retained the spectroscopic and thermodynamic properties characteristic of the corresponding domain in the native spinach enzyme.