Folding of the SAM-I riboswitch: A tale with a twist

Riboswitches are ligand-dependent RNA genetic regulators that control gene expression by altering their structures. The elucidation of riboswitch conformational changes before and after ligand recognition is crucial to understand how riboswitches can achieve high ligand binding affinity and discrimination against cellular analogs. The detailed characterization of riboswitch folding pathways suggest that they may use their intrinsic conformational dynamics to sample a large array of structures, some of which being nearly identical to ligand-bound molecules. Some of these structural conformers can be "captured" upon ligand binding, which is crucial for the outcome of gene regulation. Recent studies about the SAM-I riboswitch have revealed unexpected and previously unknown RNA folding mechanisms. For instance, the observed helical twist of the P1 stem upon ligand binding to the SAM-I aptamer adds a new element in the repertoire of RNA strategies for recognition of small metabolites. From an RNA folding perspective, these findings also strongly indicate that the SAM-I riboswitch could achieve ligand recognition by using an optimized combination of conformational capture and induced-fit approaches, a feature that may be shared by other RNA regulatory sequences.     

The yjdF riboswitch candidate regulates gene expression by binding diverse azaaromatic compounds

The yjdF motif RNA is an orphan riboswitch candidate that almost exclusively associates with the yjdF protein-coding gene in many bacteria. The function of the YjdF protein is unknown, which has made speculation regarding the natural ligand for this putative riboswitch unusually challenging. By using a structure-probing assay for ligand binding, we found that a surprisingly broad diversity of nitrogen-containing aromatic heterocycles, or “azaaromatics,” trigger near-identical changes in the structures adopted by representative yjdF motif RNAs. Regions of the RNA that undergo ligand-induced structural modulation reside primarily in portions of the putative aptamer region that are highly conserved in nucleotide sequence, as is typical for riboswitches. Some azaaromatic molecules are bound by the RNA with nanomolar dissociation constants, and a subset of these ligands activate riboswitch-mediated gene expression in cells. Furthermore, genetic elements most commonly adjacent to the yjdF motif RNA or to the yjdF protein-coding region are homologous to protein regulators implicated in mitigating the toxic effects of diverse phenolic acids or polycyclic compounds. Although the precise type of natural ligand sensed by yjdF motif RNAs remains unknown, our findings suggest that this riboswitch class might serve as part of a genetic response system to toxic or signaling compounds with chemical structures similar to azaaromatics.     

Structural principles of nucleoside selectivity in a 2'-deoxyguanosine riboswitch

Purine riboswitches have an essential role in genetic regulation of bacterial metabolism. This family includes the 2'-deoxyguanosine (dG) riboswitch, which is involved in feedback control of deoxyguanosine biosynthesis. To understand the principles that define dG selectivity, we determined crystal structures of the natural Mesoplasma florum riboswitch bound to cognate dG as well as to noncognate guanosine, deoxyguanosine monophosphate and guanosine monophosphate. Comparison with related purine riboswitch structures reveals that the dG riboswitch achieves its specificity through modification of key interactions involving the nucleobase and rearrangement of the ligand-binding pocket to accommodate the additional sugar moiety. In addition, we observe new conformational changes beyond the junctional binding pocket extending as far as peripheral loop-loop interactions. It appears that re-engineering riboswitch scaffolds will require consideration of selectivity features dispersed throughout the riboswitch tertiary fold, and structure-guided drug design efforts targeted to junctional RNA scaffolds need to be addressed within such an expanded framework.     

RNA tertiary interactions in a riboswitch stabilize the structure of a kink turn

The kink turn is a widespread RNA motif that introduces an acute kink into the axis of duplex RNA, typically comprising a bulge followed by a G?A and A?G pairs. The kinked conformation is stabilized by metal ions, or the binding of proteins including L7Ae. We now demonstrate a third mechanism for the stabilization of k-turn structure, involving tertiary interactions within a larger RNA structure. The SAM-I riboswitch contains an essential standard k-turn sequence that kinks a helix so that its terminal loop can make?a?long-range interaction. We find that some sequence variations in the k-turn within the riboswitch do not prevent SAM binding, despite preventing the folding of the k-turn in isolation. Furthermore, two crystal structures show that the sequence-variant k-turns are conventionally folded within the riboswitch. This study shows that the folded structure of the k-turn can be stabilized by tertiary interactions within a larger RNA structure.     

Riboswitches as antibacterial drug targets

New validated cellular targets are needed to reinvigorate antibacterial drug discovery. This need could potentially be filled by riboswitches-messenger RNA (mRNA) structures that regulate gene expression in bacteria. Riboswitches are unique among RNAs that serve as drug targets in that they have evolved to form structured and highly selective receptors for small drug-like metabolites. In most cases, metabolite binding to the receptor represses the expression of the gene(s) encoded by the mRNA. If a new metabolite analog were designed that binds to the receptor, the gene(s) regulated by that riboswitch could be repressed, with a potentially lethal effect to the bacteria. Recent work suggests that certain antibacterial compounds discovered decades ago function at least in part by targeting riboswitches. Herein we will summarize the experiments validating riboswitches as drug targets, describe the existing technology for riboswitch drug discovery and discuss the challenges that may face riboswitch drug discoverers.     

Searching genomes for ribozymes and riboswitches

New regulatory RNAs with complex structures have recently been discovered, among them the first catalytic riboswitch, a gene-regulatory RNA sequence with catalytic activity. Here we discuss some of the experimental approaches and theoretical difficulties attached to the identification of new ribozymes in genomes.     

核开关结构、机制及应用新进展

核开关(riboswitch)是Breaker等在2002年发现的一种全新的转录后调节机制．它可以通过小分子与mRNA结合来直接调控基因的表达,不需要任何蛋白质的参与．与常见的经由蛋白质的调控方式相比,riboswitch响应更迅速,对细胞内代谢物的变化更敏感．它的发现为RNA研究展示了新的领域． 目前在这个领域,既有基础研究,如riboswitch晶体结构解析、作用机制和动力学研究,又有前沿应用研究,如基于riboswitch的生物传感器和药物设计．Topp等通过设计riboswitch成功地改变了大肠杆菌的趋化性,这为合成生物学和人工生物网络的设计提供了新思路．目前对于riboswitch结构、机制及动力学的研究为基于riboswitch的合理药物设计奠定了基础,有望针对这一新的机制开发新一代抗菌药物．     

Single-molecule studies of riboswitch folding

The folding dynamics of riboswitches are central to their ability to modulate gene expression in response to environmental cues. In most cases, a structural competition between the formation of a ligand-binding aptamer and an expression platform (or some other competing off-state) determines the regulatory outcome. Here, we review single-molecule studies of riboswitch folding and function, predominantly carried out using single-molecule FRET or optical trapping approaches. Recent results have supplied new insights into riboswitch folding energy landscapes, the mechanisms of ligand binding, the roles played by divalent ions, the applicability of hierarchical folding models, and kinetic vs. thermodynamic control schemes. We anticipate that future work, based on improved data sets and potentially combining multiple experimental techniques, will enable the development of more complete models for complex RNA folding processes. This article is part of a Special Issue entitled: Riboswitches.     

Riboswitch regulation mechanisms: RNA,metabolites and regulatory proteins

Riboswitches are RNA sensors that have been shown to modulate the expression of downstream genes by altering their structure upon metabolite binding. Riboswitches are unique among cellular regulators in that metabolite detection is strictly performed using RNA interactions with the sensed metabolite and in which no regulatory protein is needed to mediate the interaction. However, recent studies have shed light on riboswitch control mechanisms relying on protein regulators to harness metabolite binding for the mediation of gene expression, thereby increasing the range of cellular factors involved in riboswitch regulation. The interaction between riboswitches and proteins adds another level of evolutionary pressure as riboswitches must maintain key residues for metabolite detection, structural switching and protein binding sites. Here, we review regulatory mechanisms involving Escherichia coli riboswitches that have recently been shown to rely on regulatory proteins. We also discuss the implication of such protein-based riboswitch regulatory mechanisms for genetic regulation.     

Riboswitches in unexpected places--a synthetic riboswitch in a protein coding region

In natural and engineered systems, cis-RNA regulatory elements such as riboswitches are typically found within untranslated regions rather than within the protein coding sequences of genes. However, RNA sequences with important regulatory roles can exist within translated regions. Here, we present a synthetic riboswitch that is encoded within the translated region of a gene and represses Escherichia coli gene expression greater than 25-fold in the presence of a small-molecule ligand. The ability to encode riboswitches within translated regions as well as untranslated regions provides additional opportunities for creating new genetic control elements. Furthermore, evidence that a riboswitch can function in the translated region of a gene suggests that future efforts to identify natural riboswitches should consider this possibility.     

一种新发现的基因表达调控机制——核糖开关

最近发现 ,某些依赖代谢物调节的基因转录产物的 5′UTR存在特征性结构———核糖开关(riboswitch) .核糖开关可以特异性结合代谢物 ,通过构象变化 ,在转录或翻译水平上调节基因表达 .核糖开关广泛存在于G+ 及G-细菌的代谢相关基因中 ,在真菌、植物中也有发现 .核糖开关调节维生素、氨基酸、核苷酸等基础代谢过程 ,其调节基因表达不需要任何蛋白因子作为中介 ,在进化上可能是RNA世界遗留的分子化石 .核糖开关可用于研究基因功能 ,开发新型药物及基因治疗 .     

核糖核酸调节子的构建与即时检测中的应用*

合成生物学专注于可重复利用模块和元件的工程化设计,并在生物系统中表现出良好的行为和功能。在无细胞蛋白表达系统中,核糖核酸调节子作为即时检测中的重要传感元件,通过靶标分子的诱导使其自身结构发生变化,进而调控下游基因的表达。系统介绍不同类型的核糖核酸调节子及其作用原理,包括一代核糖核酸调节子、toehold开关、功能拓展的核糖核酸调节子和核糖开关。详细阐明构建核糖核酸调节子的设计-测试-分析过程:计算机辅助设计、基因表达测试和结构功能化分析。汇总基于核糖核酸调节子的体外即时检测应用,重点总结toehold开关介导的病原菌核酸检测和核糖开关参与的小分子检测。讨论当前无细胞即时检测的特点、挑战和发展趋势,为开发新型核糖核酸调节子和即时检测工具提供思路和参考。