Faculty Bibliography

Riboswitches specifically control expression of genes predominantly involved in biosynthesis, catabolism and transport of various cellular metabolites in organisms from all three kingdoms of life. Among many classes of identified riboswitches, two riboswitches respond to amino acids lysine and glycine to date. Though these riboswitches recognize small compounds, they both belong to the largest riboswitches and have unique structural and functional characteristics. In this review, we attempt to characterize molecular recognition principles employed by amino acid-responsive riboswitches to selectively bind their cognate ligands and to effectively perform a gene regulation function. We summarize up-to-date biochemical and genetic data available for the lysine and glycine riboswitches and correlate these results with recent high-resolution structural information obtained for the lysine riboswitch. We also discuss the contribution of lysine riboswitches to antibiotic resistance and outline potential applications of riboswitches in biotechnology and medicine

Regulatory mRNA elements or riboswitches specifically control the expression of a large number of genes in response to various cellular metabolites. The basis for selectivity of regulation is programmed in the evolutionarily conserved metabolite-sensing regions of riboswitches, which display a plethora of sequence and structural variants. Recent X-ray structures of two distinct SAM riboswitches and the sensing domains of the Mg(2+), lysine, and FMN riboswitches have uncovered novel recognition principles and provided molecular details underlying the exquisite specificity of metabolite binding by RNA. These and earlier structures constitute the majority of widespread riboswitch classes and, together with riboswitch folding studies, improve our understanding of the mechanistic principles involved in riboswitch-mediated gene expression control

Site-specific 2'-methylseleno RNA labeling is a promising tool for tackling the phase problem in RNA crystallography. We have developed an efficient strategy for crystallization and structure determination of RNA and RNA/protein complexes based on preliminary crystallization screening of 2'-OCH(3)-modified RNA sequences, prior to the replacement of 2'-OCH(3) groups with their 2'-SeCH(3) counterparts. The method exploits the similar crystallization properties of 2'-OCH(3)- and 2'-SeCH(3)-modified RNAs and has been successfully validated for two test cases. In addition, our data show that 2'-SeCH(3)-modified RNA have an increased resistance to X-ray radiolysis in comparison with commonly used 5-halogen-modified RNA, which permits collection of experimental electron density maps of remarkable quality

The biosynthesis of several protein cofactors is subject to feedback regulation by riboswitches. Flavin mononucleotide (FMN)-specific riboswitches, also known as RFN elements, direct expression of bacterial genes involved in the biosynthesis and transport of riboflavin (vitamin B(2)) and related compounds. Here we present the crystal structures of the Fusobacterium nucleatum riboswitch bound to FMN, riboflavin and antibiotic roseoflavin. The FMN riboswitch structure, centred on an FMN-bound six-stem junction, does not fold by collinear stacking of adjacent helices, typical for folding of large RNAs. Rather, it adopts a butterfly-like scaffold, stapled together by opposingly directed but nearly identically folded peripheral domains. FMN is positioned asymmetrically within the junctional site and is specifically bound to RNA through interactions with the isoalloxazine ring chromophore and direct and Mg(2+)-mediated contacts with the phosphate moiety. Our structural data, complemented by binding and footprinting experiments, imply a largely pre-folded tertiary RNA architecture and FMN recognition mediated by conformational transitions within the junctional binding pocket. The inherent plasticity of the FMN-binding pocket and the availability of large openings make the riboswitch an attractive target for structure-based design of FMN-like antimicrobial compounds. Our studies also explain the effects of spontaneous and antibiotic-induced deregulatory mutations and provided molecular insights into FMN-based control of gene expression in normal and riboflavin-overproducing bacterial strains

Riboswitches are mRNA regions that regulate the expression of genes in response to various cellular metabolites. These RNA sequences, typically situated in the untranslated regions of mRNAs, possess complex structures that dictate highly specific binding to certain ligands, such as nucleobases, coenzymes, amino acids, and sugars, without protein assistance. Depending on the presence of the ligand, metabolite-binding domains of riboswitches can adopt two alternative conformations, which define the conformations of the adjacent sequences involved in the regulation of gene expression. In order to understand in detail the nature of riboswitch-ligand interactions and the molecular basis of riboswitch-based gene expression control, it is necessary to determine the three-dimensional structures of riboswitch-ligand complexes. This chapter outlines the techniques that are employed to prepare riboswitch-ligand complexes for structure determination using X-ray crystallography. The chapter describes the principles of construct design, in vitro transcription, RNA purification, complex formation, and crystallization screening utilized during the successful crystallization of several riboswitches

In bacteria, the intracellular concentration of several amino acids is controlled by riboswitches. One of the important regulatory circuits involves lysine-specific riboswitches, which direct the biosynthesis and transport of lysine and precursors common for lysine and other amino acids. To understand the molecular basis of amino acid recognition by riboswitches, here we present the crystal structure of the 174-nucleotide sensing domain of the Thermotoga maritima lysine riboswitch in the lysine-bound (1.9 angstrom (A)) and free (3.1 A) states. The riboswitch features an unusual and intricate architecture, involving three-helical and two-helical bundles connected by a compact five-helical junction and stabilized by various long-range tertiary interactions. Lysine interacts with the junctional core of the riboswitch and is specifically recognized through shape-complementarity within the elongated binding pocket and through several direct and K(+)-mediated hydrogen bonds to its charged ends. Our structural and biochemical studies indicate preformation of the riboswitch scaffold and identify conformational changes associated with the formation of a stable lysine-bound state, which prevents alternative folding of the riboswitch and facilitates formation of downstream regulatory elements. We have also determined several structures of the riboswitch bound to different lysine analogues, including antibiotics, in an effort to understand the ligand-binding capabilities of the lysine riboswitch and understand the nature of antibiotic resistance. Our results provide insights into a mechanism of lysine-riboswitch-dependent gene control at the molecular level, thereby contributing to continuing efforts at exploration of the pharmaceutical and biotechnological potential of riboswitches

Messenger RNAs interact with a number of different molecules that determine the fate of each transcript and contribute to the overall pattern of gene expression. These interactions are governed by specific mRNA signals, which in principle could represent a special mRNA recognition 'code'. Both, small molecules and proteins demonstrate a diversity of mRNA binding modes often dependent on the structural context of the regions surrounding specific target sequences. In this review, we have highlighted recent structural studies that illustrate the diversity of recognition principles used by mRNA binders for timely and specific targeting and processing of the message

Although various functions of RNA are carried out in conjunction with proteins, some catalytic RNAs, or ribozymes, which contribute to a range of cellular processes, require little or no assistance from proteins. Furthermore, the discovery of metabolite-sensing riboswitches and other types of RNA sensors has revealed RNA-based mechanisms that cells use to regulate gene expression in response to internal and external changes. Structural studies have shown how these RNAs can carry out a range of functions. In addition, the contribution of ribozymes and riboswitches to gene expression is being revealed as far more widespread than was previously appreciated. These findings have implications for understanding how cellular functions might have evolved from RNA-based origins

Gene expression can be regulated at the level of initiation of protein biosynthesis via structural elements present at the 5' untranslated region of mRNAs. These folded mRNA segments may bind to the ribosome, thus blocking translation until the mRNA unfolds. Here, we report a series of cryo-electron microscopy snapshots of ribosomal complexes directly visualizing either the mRNA structure blocked by repressor protein S15 or the unfolded, active mRNA. In the stalled state, the folded mRNA prevents the start codon from reaching the peptidyl-tRNA (P) site inside the ribosome. Upon repressor release, the mRNA unfolds and moves into the mRNA channel allowing translation initiation. A comparative structure and sequence analysis suggests the existence of a universal stand-by site on the ribosome (the 30S platform) dedicated for binding regulatory 5' mRNA elements. Different types of mRNA structures may be accommodated during translation preinitiation and regulate gene expression by transiently stalling the ribosome