Comparative molecular dynamics simulation analysis of G20 and C92 mutations in c-di-GMP I riboswitch and the wild type with docked c-di-GMP ligand

Riboswitch, a bacterial regulatory RNA consists of an aptamer (specific ligand binding unit) and an expression platform (gene expression modulation unit), which act as a potential drug target as it regulates critical genes. Therefore, it is of interest to glean information on the binding of c-di-GMP ligand to mutated conserved G20 and C92 residues of cyclic diguanosine monophosphate I (c-di-GMP I) riboswitch using molecular dynamics simulation. The result shows that the binding energy of wild/native type riboswitch-ligand complex (3IRW) is lower than the mutant complexes suggesting that the binding affinity for c-di-GMP ligand decreases in case of mutant riboswitches. The hydrogen bonding interactions analysis also showed a high number of hydrogen bonds formation in the wild type riboswitch-ligand complex as compared to the mutant complexes illustrating stronger interaction of ligand to wild type riboswitch than the mutants. The simulation result shows that the mutations affected riboswitch-ligand interactions. The residues G14, G21, C46, A47, and U92 were identified as the key residues which contributed effectively to the binding of c-di-GMP I riboswitch with the natural ligand.     

Structural and biochemical characterization of linear dinucleotide analogues bound to the c-di-GMP-I aptamer

The cyclic dinucleotide c-di-GMP regulates lifestyle transitions in many bacteria, such as the change from a free motile state to a biofilm-forming community. Riboswitches that bind this second messenger are important downstream targets in this bacterial signaling pathway. The breakdown of c-di-GMP in the cell is accomplished enzymatically and results in the linear dinucleotide pGpG. The c-di-GMP-binding riboswitches must be able to discriminate between their cognate cyclic ligand and linear dinucleotides in order to be selective biological switches. It has been reported that the c-di-GMP-I riboswitch binds c-di-GMP 5 orders of magnitude better than the linear pGpG, but the cause of this large energetic difference in binding is unknown. Here we report binding data and crystal structures of several linear c-di-GMP analogues in complex with the c-di-GMP-I riboswitch. These data reveal the parameters for phosphate recognition and the structural basis of linear dinucleotide binding to the riboswitch. Additionally, the pH dependence of binding shows that exclusion of pGpG is not due to the additional negative charge on the ligand. These data reveal principles that, along with published work, will contribute to the design of c-di-GMP analogues with properties desirable for use as chemical tools and potential therapeutics.     

Differential binding of 2'-biotinylated analogs of c-di-GMP with c-di-GMP riboswitches and binding proteins

C-di-GMP has emerged as a signalling molecule that regulates a variety of processes in several bacteria; therefore there is interest in the development of biotinylated analogs for the identification of binding partners. No detailed study has been done to evaluate if biotinylated analogs of c-di-GMP are capable of binding to c-di-GMP receptors. Herein, we evaluate the binding of commercially available 2'-biotinylated c-di-GMP and phosphorothioate 2'-biotinylated c-di-GMP, prepared via a facile solid-phase synthesis, to several c-di-GMP receptors. Docking, using Autodock vina software, as well as experimental studies of these analogs, with c-di-GMP class I and II riboswitches and binding proteins, reveal that some, but not all, c-di-GMP receptors can tolerate the 2'-modification of c-di-GMP with biotin.     

Riboswitch regulation in cyanobacteria is independent of their habitat adaptations

Cyanobacteria are one of the ancient bacterial species occupying a variety of habitats with diverse metabolic preferences. RNA regulators like riboswitches play significant role in controlling the gene expression in prokaryotes. The taxonomic distribution of riboswitches suggests that they might be one of the oldest mechanisms of gene control system. In this paper, we analyzed the distribution of different riboswitch families in various cyanobacterial genomes. It was observed that only four riboswitch classes were abundant in cyanobacteria, B₁₂-element (Cob)/AdoCbl/AdoCbl-variant riboswitch being the most abundant. The analysis suggests that riboswitch mode of regulation is present in cyanobacterial species irrespective of their habitat types. A large number of unidentified genes regulated by riboswitches listed in this analysis indicate the wide range of targets for these riboswitch families. The analysis revealed a large number of genes regulated by riboswitches which may assist in elaborating the diversity among the cyanobacterial species.     

The RNA Domain Vc1 Regulates Downstream Gene Expression in Response to Cyclic Diguanylate in Vibrio cholerae

In many bacterial species, including the aquatic bacterium and human pathogen Vibrio cholerae, the second messenger cyclic diguanylate (c-di-GMP) modulates processes such as biofilm formation, motility, and virulence factor production. By interacting with various effectors, c-di-GMP regulates gene expression or protein function. One type of c-di-GMP receptor is the class I riboswitch, representatives of which have been shown to bind c-di-GMP in vitro. Herein, we examined the in vitro and in vivo function of the putative class I riboswitch in Vibrio cholerae, Vc1, which lies upstream of the gene encoding GbpA, a colonization factor that contributes to attachment of V. cholerae to environmental and host surfaces containing N-acetylglucosamine moieties. We provide evidence that Vc1 RNA interacts directly with c-di-GMP in vitro, and that nucleotides conserved among this class of riboswitch are important for binding. Yet the mutation of these conserved residues individually in the V. cholerae chromosome inconsistently affects the expression of gbpA and production of the GbpA protein. By isolating the regulatory function of Vc1, we show that the Vc1 element positively regulates downstream gene expression in response to c-di-GMP. Together these data suggest that the Vc1 element responds to c-di-GMP in vivo. Positive regulation of gbpA expression by c-di-GMP via Vc1 may influence the ability of V. cholerae to associate with chitin in the aquatic environment and the host intestinal environment.     

Interactions of the c-di-GMP riboswitch with its second messenger ligand

The c-di-GMP [bis-(3'-5')-cyclic dimeric guanosine monophosphate] riboswitch is a macromolecular target in the c-di-GMP second messenger signalling pathway. It regulates many genes related to c-di-GMP metabolism as well as genes involved in bacterial motility, virulence and biofilm formation. The riboswitch makes asymmetric contacts to the bases and phosphate backbone of this symmetric dinucleotide. The phylogenetics suggested and mutagenesis has confirmed that this is a flexible motif where variants can make alternative interactions with each of the guanine bases of c-di-GMP. A mutant riboswitch has been designed that can bind a related molecule, c-di-AMP, confirming the most important contacts made to the ligand. The binding kinetics reveal that this is a kinetically controlled riboswitch and mutations to the riboswitch lead to increases in the off-rate. This riboswitch is therefore flexible in sequence as well as kinetic properties.     

Riboswitches that sense S-adenosylmethionine and S-adenosylhomocysteine.

Numerous riboswitches have been discovered that specifically recognize metabolites and modulate gene expression. Each riboswitch class is defined either by the consensus sequence and structural features of its metabolite-binding aptamer domain, or by the distinct metabolite that the aptamer recognizes. Several distinct classes of riboswitches that respond to S-adenosylmethionine (SAM or AdoMet) have been discovered. Representatives of these classes have been shown to strongly discriminate against S-adenosylhomocystenine (SAH or AdoHcy), which is the metabolic byproduct produced when SAM is used as a cofactor for methylation reactions. However, a distinct class of riboswitches that selectively binds SAH, and strongly discriminates against SAM, also has been discovered. Herein we compare the features of SAM and SAH riboswitches, which help showcase the enormous structural diversity that RNA can harness to form precision genetic switches for compounds that are critical for fundamental metabolic processes.     

Structure and mechanism of purine-binding riboswitches

A riboswitch is a non-protein coding sequence capable of directly binding a small molecule effector without the assistance of accessory proteins to regulate expression of the mRNA in which it is embedded. Currently, over 20 different classes of riboswitches have been validated in bacteria with the promise of many more to come, making them an important means of regulating the genome in the bacterial kingdom. Strikingly, half of the known riboswitches recognize effector compounds that contain a purine or related moiety. In the last decade, significant progress has been made to determine how riboswitches specifically recognize these compounds against the background of many other similar cellular metabolites and transduce this signal into a regulatory response. Of the known riboswitches, the purine family containing guanine, adenine and 2'-deoxyguanosine-binding classes are the most extensively studied, serving as a simple and useful paradigm for understanding how these regulatory RNAs function. This review provides a comprehensive summary of the current state of knowledge regarding the structure and mechanism of these riboswitches, as well as insights into how they might be exploited as therapeutic targets and novel biosensors.     

Evidence for a second class of S-adenosylmethionine riboswitches and other regulatory RNA motifs in alpha-proteobacteria

Background

Riboswitches are RNA elements in the 5' untranslated leaders of bacterial mRNAs that directly sense the levels of specific metabolites with a structurally conserved aptamer domain to regulate expression of downstream genes. Riboswitches are most common in the genomes of low GC Gram-positive bacteria (for example, Bacillus subtilis contains examples of all known riboswitches), and some riboswitch classes seem to be restricted to this group.

Results

We used comparative sequence analysis and structural probing to identify five RNA elements (serC, speF, suhB, ybhL, and metA) that reside in the intergenic regions of Agrobacterium tumefaciens and many other α-proteobacteria. One of these, the metA motif, is found upstream of methionine biosynthesis genes and binds S-adenosylmethionine (SAM). This natural aptamer most likely functions as a SAM riboswitch (SAM-II) with a consensus sequence and structure that is distinct from the class of SAM riboswitches (SAM-I) predominantly found in Gram-positive bacteria. The minimal functional SAM-II aptamer consists of fewer than 70 nucleotides, which form a single stem and a pseudoknot. Despite its simple architecture and lower affinity for SAM, the SAM-II aptamer strongly discriminates against related compounds.

Conclusion

SAM-II is the only metabolite-binding riboswitch class identified so far that is not found in Gram-positive bacteria, and its existence demonstrates that biological systems can use multiple RNA structures to sense a single chemical compound. The two SAM riboswitches might be 'RNA World' relics that were selectively retained in certain bacterial lineages or new motifs that have emerged since the divergence of the major bacterial groups.     

鉴定cyclic di-GMP效应蛋白：高通量筛选策略与实验验证方法

环二鸟苷单磷酸(cyclic di-GMP或c-di-GMP)是细菌细胞中广泛存在的第二信使,调控细菌生物被膜发育、致病力、运动性、胞外多糖产生及细胞周期在内的诸多重要生理表型。c-di-GMP通过结合多种类型的效应子(包括核糖开关或效应蛋白)来发挥调控功能。由于c-di-GMP分子在构象上具有多变性,其结合的效应子同样具有多样性。新型效应蛋白的筛选、鉴定是当前细菌信号转导领域的研究热点和难点,也是解析c-di-GMP调控机制的首要环节。本文在阐述c-di-GMP结合不同类型的效应蛋白并调控细菌生物被膜发育的基础上,综述了目前筛选c-di-GMP效应蛋白的方法,包括遗传筛选、亲和色谱结合质谱鉴定、DRa CALA系统鉴定以及基于分子对接的预测等。同时,对验证c-di-GMP效应蛋白的技术,如等温微量热滴定、表面等离子共振、微量热泳动在内的多种验证方法进行了总结,对比了这些策略和方法在应用上的优、缺点,为在细菌及其真核宿主基因组水平鉴定c-di-GMP效应蛋白的研究提供参考。     

High-throughput screening of cell-free riboswitches by fluorescence-activated droplet sorting

Cell-free systems that display complex functions without using living cells are emerging as new platforms to test our understanding of biological systems as well as for practical applications such as biosensors and biomanufacturing. Those that use cell-free protein synthesis (CFPS) systems to enable genetically programmed protein synthesis have relied on genetic regulatory components found or engineered in living cells. However, biological constraints such as cell permeability, metabolic stability, and toxicity of signaling molecules prevent development of cell-free devices using living cells even if cell-free systems are not subject to such constraints. Efforts to engineer regulatory components directly in CFPS systems thus far have been based on low-throughput experimental approaches, limiting the availability of basic components to build cell-free systems with diverse functions. Here, we report a high-throughput screening method to engineer cell-free riboswitches that respond to small molecules. Droplet-sorting of riboswitch variants in a CFPS system rapidly identified cell-free riboswitches that respond to compounds that are not amenable to bacterial screening methods. Finally, we used a histamine riboswitch to demonstrate chemical communication between cell-sized droplets.     

Evolutionary Evidence for Alternative Structure in RNA Sequence Co-variation

Sequence conservation and co-variation of base pairs are hallmarks of structured RNAs. For certain RNAs (e.g. riboswitches), a single sequence must adopt at least two alternative secondary structures to effectively regulate the message. If alternative secondary structures are important to the function of an RNA, we expect to observe evolutionary co-variation supporting multiple conformations. We set out to characterize the evolutionary co-variation supporting alternative conformations in riboswitches to determine the extent to which alternative secondary structures are conserved. We found strong co-variation support for the terminator, P1, and anti-terminator stems in the purine riboswitch by extending alignments to include terminator sequences. When we performed Boltzmann suboptimal sampling on purine riboswitch sequences with terminators we found that these sequences appear to have evolved to favor specific alternative conformations. We extended our analysis of co-variation to classic alignments of group I/II introns, tRNA, and other classes of riboswitches. In a majority of these RNAs, we found evolutionary evidence for alternative conformations that are compatible with the Boltzmann suboptimal ensemble. Our analyses suggest that alternative conformations are selected for and thus likely play functional roles in even the most structured of RNAs.     

Purine sensing by riboswitches.

Structured mRNA elements called riboswitches control gene expression by binding to small metabolites. Over a dozen riboswitch classes have been characterized that target a broad range of molecules and vary widely in size and secondary structure. Four of the known riboswitch classes recognize purines or modified purines. Three of these classes are closely related in conserved sequence and secondary structure, but members of these classes selectively recognize guanine, adenine or 2'-deoxyguanosine. Members of the fourth riboswitch class adopt a distinct structure to form a selective binding pocket for the guanine analogue preQ(1) (7-aminomethyl-7-deazaguanine). All four classes of purine-sensing riboswitches are most likely to recognize their respective metabolites by utilizing a riboswitch residue to make a canonical Watson-Crick base-pair with the ligand. This review will provide a summary of the purine-sensing riboswitches, as well as discuss the complex functions and applications of these RNAs.