环二鸟苷单磷酸核糖开关的结构与功能

环二鸟苷单磷酸(c-di-GMP)是细菌中广泛存在的一类核苷类第二信使分子,能够调控细菌的生物被膜形成、运动性、黏附、毒力以及胞外多糖的产生等众多生理活动。核糖开关是m RNA 5′-非翻译区(5′-Untranslational region,5′-UTR)的一段RNA序列,包含可以识别并结合配体的保守序列——适配体区(Aptamer domain,AD),以及结构多变、可以调控下游编码基因的表达平台区(Expression platform,EP)。当代谢物分子浓度比较高时,其与适配体区结合,引起下游的表达平台区发生构象变化,进而实现对下游基因的调节。目前已发现c-di-GMP-Ⅰ和c-di-GMP-Ⅱ两类c-di-GMP的核糖开关。它们通过特异性地结合c-di-GMP,调控种类繁多的下游基因的表达。c-di-GMP-I核糖开关分布广泛,尤其在厚壁菌门(Firmicutes)和变形菌门(Proteobacteria)的细菌中最为丰富。c-di-GMP-Ⅱ核糖开关具备变构核酶的功能,结合c-di-GMP后在其非典型剪切位点处发生结构变化,调节下游基因表达。文中围绕c-di-GMP核糖开关的发现、功能、分类以及下游调控基因的功能进行综述与分析。     

A kinetic and thermodynamic framework for the Azoarcus group I ribozyme reaction

Determination of quantitative thermodynamic and kinetic frameworks for ribozymes derived from the Azoarcus group I intron and comparisons to their well-studied analogs from the Tetrahymena group I intron reveal similarities and differences between these RNAs. The guanosine (G) substrate binds to the Azoarcus and Tetrahymena ribozymes with similar equilibrium binding constants and similar very slow association rate constants. These and additional literature observations support a model in which the free ribozyme is not conformationally competent to bind G and in which the probability of assuming the binding-competent state is determined by tertiary interactions of peripheral elements. As proposed previously, the slow binding of guanosine may play a role in the specificity of group I intron self-splicing, and slow binding may be used analogously in other biological processes. The internal equilibrium between ribozyme-bound substrates and products is similar for these ribozymes, but the Azoarcus ribozyme does not display the coupling in the binding of substrates that is observed with the Tetrahymena ribozyme, suggesting that local preorganization of the active site and rearrangements within the active site upon substrate binding are different for these ribozymes. Our results also confirm the much greater tertiary binding energy of the 5′-splice site analog with the Azoarcus ribozyme, binding energy that presumably compensates for the fewer base-pairing interactions to allow the 5′-exon intermediate in self splicing to remain bound subsequent to 5′-exon cleavage and prior to exon ligation. Most generally, these frameworks provide a foundation for design and interpretation of experiments investigating fundamental properties of these and other structured RNAs.     

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.     

An active site rearrangement within the Tetrahymena group I ribozyme releases nonproductive interactions and allows formation of catalytic interactions

Biological catalysis hinges on the precise structural integrity of an active site that binds and transforms its substrates and meeting this requirement presents a unique challenge for RNA enzymes. Functional RNAs, including ribozymes, fold into their active conformations within rugged energy landscapes that often contain misfolded conformers. Here we uncover and characterize one such “off-pathway” species within an active site after overall folding of the ribozyme is complete. The Tetrahymena group I ribozyme (E) catalyzes cleavage of an oligonucleotide substrate (S) by an exogenous guanosine (G) cofactor. We tested whether specific catalytic interactions with G are present in the preceding E•S•G and E•G ground-state complexes. We monitored interactions with G via the effects of 2′- and 3′-deoxy (–H) and −amino (–NH₂) substitutions on G binding. These and prior results reveal that G is bound in an inactive configuration within E•G, with the nucleophilic 3′-OH making a nonproductive interaction with an active site metal ion termed M_A and with the adjacent 2′-OH making no interaction. Upon S binding, a rearrangement occurs that allows both –OH groups to contact a different active site metal ion, termed M_C, to make what are likely to be their catalytic interactions. The reactive phosphoryl group on S promotes this change, presumably by repositioning the metal ions with respect to G. This conformational transition demonstrates local rearrangements within an otherwise folded RNA, underscoring RNA''s difficulty in specifying a unique conformation and highlighting Nature''s potential to use local transitions of RNA in complex function.     

RNA tertiary interactions mediate native collapse of a bacterial group I ribozyme

Large RNAs collapse into compact intermediates in the presence of counterions before folding to the native state. We previously found that collapse of a bacterial group I ribozyme correlates with the formation of helices within the ribozyme core, but occurs at Mg2+ concentrations too low to support stable tertiary structure and catalytic activity. Here, using small-angle X-ray scattering, we show that Mg2+-induced collapse is a cooperative folding transition that can be fit by a two-state model. The Mg2+ dependence of collapse is similar to the Mg2+ dependence of helix assembly measured by partial ribonuclease T1 digestion and of an unfolding transition measured by UV hypochromicity. The correspondence between multiple probes of RNA structure further supports a two-state model. A mutation that disrupts tertiary contacts between the L9 tetraloop and its helical receptor destabilized the compact state by 0.8 kcal/mol, while mutations in the central triplex were less destabilizing. These results show that native tertiary interactions stabilize the compact folding intermediates under conditions in which the RNA backbone remains accessible to solvent.     

The Azoarcus group I intron ribozyme misfolds and is accelerated for refolding by ATP-dependent RNA chaperone proteins

Structured RNAs traverse complex energy landscapes that include valleys representing misfolded intermediates. In Neurospora crassa and Saccharomyces cerevisiae, efficient splicing of mitochondrial group I and II introns requires the DEAD box proteins CYT-19 and Mss116p, respectively, which promote folding transitions and function as general RNA chaperones. To test the generality of RNA misfolding and the activities of DEAD box proteins in vitro, here we measure native folding of a small group I intron ribozyme from the bacterium Azoarcus by monitoring its catalytic activity. To develop this assay, we first measure cleavage of an oligonucleotide substrate by the prefolded ribozyme. Substrate cleavage is rate-limited by binding and is readily reversible, with an internal equilibrium near unity, such that the amount of product observed is less than the amount of native ribozyme. We use this assay to show that approximately half of the ribozyme folds readily to the native state, whereas the other half forms an intermediate that transitions slowly to the native state. This folding transition is accelerated by urea and increased temperature and slowed by increased Mg(2+) concentration, suggesting that the intermediate is misfolded and must undergo transient unfolding during refolding to the native state. CYT-19 and Mss116p accelerate refolding in an ATP-dependent manner, presumably by disrupting structure in the intermediate. These results highlight the tendency of RNAs to misfold, underscore the roles of CYT-19 and Mss116p as general RNA chaperones, and identify a refolding transition for further dissection of the roles of DEAD box proteins in RNA folding.     

Toward predicting self-splicing and protein-facilitated splicing of group I introns

In the current era of massive discoveries of noncoding RNAs within genomes, being able to infer a function from a nucleotide sequence is of paramount interest. Although studies of individual group I introns have identified self-splicing and nonself-splicing examples, there is no overall understanding of the prevalence of self-splicing or the factors that determine it among the >2300 group I introns sequenced to date. Here, the self-splicing activities of 12 group I introns from various organisms were assayed under six reaction conditions that had been shown previously to promote RNA catalysis for different RNAs. Besides revealing that assessing self-splicing under only one condition can be misleading, this survey emphasizes that in vitro self-splicing efficiency is correlated with the GC content of the intron (>35% GC was generally conductive to self-splicing), and with the ability of the introns to form particular tertiary interactions. Addition of the Neurospora crassa CYT-18 protein activated splicing of two nonself-splicing introns, but inhibited the second step of self-splicing for two others. Together, correlations between sequence, predicted structure and splicing begin to establish rules that should facilitate our ability to predict the self-splicing activity of any group I intron from its sequence.     

New insights into Legionella pneumophila biofilm regulation by c-di-GMP signaling

The waterborne pathogen Legionella pneumophila grows as a biofilm, freely or inside amoebae. Cyclic-di-GMP (c-di-GMP), a bacterial second messenger frequently implicated in biofilm formation, is synthesized and degraded by diguanylate cyclases (DGCs) and phosphodiesterases (PDEs), respectively. To characterize the c-di-GMP-metabolizing enzymes involved in L. pneumophila biofilm regulation, the consequences on biofilm formation and the c-di-GMP concentration of each corresponding gene inactivation were assessed in the Lens strain. The results showed that one DGC and two PDEs enhance different aspects of biofilm formation, while two proteins with dual activity (DGC/PDE) inhibit biofilm growth. Surprisingly, only two mutants exhibited a change in global c-di-GMP concentration. This study highlights that specific c-di-GMP pathways control L. pneumophila biofilm formation, most likely via temporary and/or local modulation of c-di-GMP concentration. Furthermore, Lpl1054 DGC is required to enable the formation a dense biofilm in response to nitric oxide, a signal for biofilm dispersion in many other species.     

Accommodation of Ca(II) ions for catalytic activity by a group I ribozyme

The wildtype Tetrahymena ribozyme cannot catalyze detectable levels of phosphotransfer activity in vitro on an exogenous RNA substrate oligonucleotide when calcium(II) is supplied as the only available divalent ion. Nevertheless, low-error mutants of this ribozyme have been acquired through directed evolution that do have activity in 10 mM CaCl₂. The mechanisms for such Ca(II) accommodation are not known. Here, we assayed the entire molecule in an effort to identify the roles of the mutations in allowing catalytic activity in Ca(II). We used four biochemical probing techniques - native-gel electrophoresis, hydroxyl radical footprinting, terbium(III) cleavage footprinting, and phosphorothioate interference mapping - to compare the solution structure of the wildtype ribozyme with that of a Ca(II)-active five-site mutant. We compared the gross folding patterns and specific metal-binding sites in both MgCl₂ and CaCl₂ solutions. We detected no large-scale folding differences between the two RNAs in either metal. However, we did discover a limited number of local folding differences, involving regions of the RNA affected by positions 42, 188, and 270. These data support the notion that Ca(II) is accommodated by the Tetrahymena ribozyme by a slight breathing at the active site, but that alterations at, near to, and distal from the active site can all contribute to Ca(II)-based activity.     

Identification of messenger and long noncoding RNAs associated with gallbladder cancer via gene expression profile analysis

The present study aimed to investigate the long noncoding RNAs (lncRNAs) and messenger RNAs (mRNAs) involved in the progression of gallbladder cancer and explore the potential physiopathologic mechanisms of gallbladder cancer in terms of competing endogenous RNAs (ceRNAs). The original lncRNA and mRNA expression profile data (nine gallbladder cancer tissues samples and nine normal gallbladder samples) in GSE76633 was downloaded from the Gene Expression Omnibus database. Differentially expressed mRNAs and lncRNAs between gallbladder cancer tissue and normal control were selected and the pathways in which they are involved were analyzed using bioinformatics analyses. MicroRNAs (miRNAs) were also predicted based on the differentially expressed mRNAs. Finally, the co-expression relation between lncRNA and mRNA was analyzed and the ceRNA network was constructed by combining the lncRNA-miRNA, miRNA-mRNA, and lncRNA-mRNA pairs. Overall, 373 significantly differentially expressed mRNAs and 47 lncRNAs were identified between cancer and normal tissue samples. The upregulated genes were significantly enriched in the extracellular matrix (ECM)-receptor interaction pathway, while the downregulated genes were involved in the complement and coagulation cascades. Altogether, 128 co-expression relations between lncRNA and mRNA were obtained. In addition, 196 miRNA-mRNA regulatory relations and 145 miRNA-lncRNA relation pairs were predicted. Finally, the lncRNA-miRNA-gene ceRNA network was constructed by combining the three types of relation pairs, such as XLOC_011309-miR-548c-3p-SPOCK1 and XLOC_012588-miR-765-CEACAM6. mRNAs and lncRNAs may be involved in gallbladder cancer progression via ECM-receptor interaction pathways and the complement and coagulation cascades. Moreover, ceRNAs such as XLOC_011309-miR-548c-3p-SPOCK1 and XLOC_012588-miR-765-CEACAM6 can also be implicated in the pathogenesis of gallbladder cancer.     

Confirmation of a second natural preQ1 aptamer class in Streptococcaceae bacteria

Bioinformatics searches of eubacterial genomes have yielded many riboswitch candidates where the identity of the ligand is not immediately obvious on examination of associated genes. One of these motifs is found exclusively in the family Streptococcaceae within the 5' untranslated regions (UTRs) of genes encoding the hypothetical membrane protein classified as COG4708 or DUF988. While the function of this protein class is unproven, a riboswitch binding the queuosine biosynthetic intermediate pre-queuosine(1) (preQ(1)) has been identified in the 5' UTR of homologous genes in many Firmicute species of bacteria outside of Streptococcaceae. Here we show that a representative of the COG4708 RNA motif from Streptococcus pneumoniae R6 also binds preQ(1). Furthermore, representatives of this RNA have structural and molecular recognition characteristics that are distinct from those of the previously described preQ(1) riboswitch class. PreQ(1) is the second metabolite for which two or more distinct classes of natural aptamers exist, indicating that natural aptamers utilizing different structures to bind the same metabolite may be more common than is currently known. Additionally, the association of preQ(1) binding RNAs with most genes encoding proteins classified as COG4708 strongly suggests that these proteins function as transporters for preQ(1) or another queuosine biosynthetic intermediate.     

Relationship between the self-splicing activity and the solidity of the master domain of the Tetrahymena group I ribozyme

The highly conserved P3-P7 domain of the Group I intron ribozymes is known to contain essential elements, such as the binding site for the cofactor guanosine, required for conducting the splicing reaction. We investigated the domain of the Tetrahymena intron ribozyme and its variants in order to clarify the relationship between its stability and function. We found that the destabilization of the P3-P7 domain facilitates the active structure formation at high magnesium ion concentrations where the formation is retarded for the wild type. The destabilized domain also increases K(GTP)(m) although this can be compensated by increasing the concentration of Mg(2+), indicating that the stable domain is required for establishing a tight guanosine binding site. The results suggest that the stability of the domain affects the rate-limiting step in the RNA folding pathway and also regulates the efficiency of the splicing reaction.     

c-di-GMP Arms an Anti-σ to Control Progression of Multicellular Differentiation in Streptomyces

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Evidence for a major role of antisense RNAs in cyanobacterial gene regulation

Information on the numbers and functions of naturally occurring antisense RNAs (asRNAs) in eubacteria has thus far remained incomplete. Here, we screened the model cyanobacterium Synechocystis sp. PCC 6803 for asRNAs using four different methods. In the final data set, the number of known noncoding RNAs rose from 6 earlier identified to 60 and of asRNAs from 1 to 73 (28 were verified using at least three methods). Among these, there are many asRNAs to housekeeping, regulatory or metabolic genes, as well as to genes encoding electron transport proteins. Transferring cultures to high light, carbon‐limited conditions or darkness influenced the expression levels of several asRNAs, suggesting their functional relevance. Examples include the asRNA to rpl1, which accumulates in a light‐dependent manner and may be required for processing the L11 r‐operon and the SyR7 noncoding RNA, which is antisense to the murF 5′ UTR, possibly modulating murein biosynthesis. Extrapolated to the whole genome, ～10% of all genes in Synechocystis are influenced by asRNAs. Thus, chromosomally encoded asRNAs may have an important function in eubacterial regulatory networks.     

Structural metals in the group I intron: a ribozyme with a multiple metal ion core

Metal ions play key roles in the folding and function for many structured RNAs, including group I introns. We determined the X-ray crystal structure of the Azoarcus bacterial group I intron in complex with its 5' and 3' exons. In addition to 222 nucleotides of RNA, the model includes 18 Mg(2+) and K(+) ions. Five of the metals bind within 12 A of the scissile phosphate and coordinate the majority of the oxygen atoms biochemically implicated in conserved metal-RNA interactions. The metals are buried deep within the structure and form a multiple metal ion core that is critical to group I intron structure and function. Eight metal ions bind in other conserved regions of the intron structure, and the remaining five interact with peripheral structural elements. Each of the 18 metals mediates tertiary interactions, facilitates local bends in the sugar-phosphate backbone or binds in the major groove of helices. The group I intron has a rich history of biochemical efforts aimed to identify RNA-metal ion interactions. The structural data are correlated to the biochemical results to further understand the role of metal ions in group I intron structure and function.     

Novel RNA polymerization reaction catalyzed by a group I ribozyme.

We have converted a bacterial tRNA precursor containing a 205 nt self-splicing group I intron into a RNA enzyme that catalyzes polymerization of an external RNA substrate. The reaction involves transesterification steps analogous to both the forward and reverse exon ligation steps of group I splicing; as such it depends entirely on 3' splice site reactions. The RNA substrate is a 20 nt analogue of the ligated exons (E1.E2), whose 3' end resembles the 3' terminus of the intron RNA enzyme (IVS). The splice junction of the substrate is attacked by the 3' end of the intron, then the molecule displaces the original 3' terminal guanosine so that the new 3' terminus is brought into the active site and used as the attacking nucleophile in the next reaction. Polymerization occurs via a series of covalent enzyme-linked intermediates of the structure IVS.(E2)n, where n = 1 to > or = 18. The 5' exon accumulates during the course of the reaction and can attack the covalent intermediates to produce elongation products of structure E1.(E2)n, regenerating the intron RNA enzyme in unchanged form. In this manner, the enzyme converts 20 nt oligoribonucleotides into polyribonucleotides up to at least 180 nt by 10 nt increments. These results have significant implications for the evolution of RNA-based self-replicating systems.