首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 15 毫秒
1.
2.
Riboswitches are cis-acting RNA fragments that regulate gene expression by sensing cellular levels of the associated small metabolites. In bacteria, the class I preQ(1) riboswitch allows the fine-tuning of queuosine biosynthesis in response to the intracellular concentration of the queuosine anabolic intermediate preQ(1). When binding preQ(1), the aptamer domain undergoes a significant degree of secondary and tertiary structural rearrangement and folds into an H-type pseudoknot. Conformational "switching" of the riboswitch aptamer domain upon recognizing its cognate metabolite plays a key role in the regulatory mechanism of the preQ(1) riboswitch. We investigate the folding mechanism of the preQ(1) riboswitch aptamer domain using all-atom Go?-model simulations. The folding pathway of such a single domain is found to be cooperative and sequentially coordinated, as the folding proceeds in the 5' → 3' direction. This kinetically efficient folding mechanism suggests a fast ligand-binding response in competition with RNA elongation.  相似文献   

3.
In a recent issue of Science, Greenleaf et al. (2008) report single-molecule force measurements to explore the sequential folding landscape of an adenine riboswitch aptamer domain. This study provides an exceptionally quantitative view of how an RNA molecule folds.  相似文献   

4.
Riboswitch‐mediated control of gene expression depends on ligand binding properties (kinetics and affinity) of its aptamer domain. A detailed analysis of interior regions of the aptamer, which affect the ligand binding properties, is important for both understanding natural riboswitch functions and for enabling rational design of tuneable artificial riboswitches. Kinetic analyses of binding reaction between flavin mononucleotide (FMN) and several natural and mutant aptamer domains of FMN‐specific riboswitches were performed. The strong dependence of the dissociation rate (52.6‐fold) and affinity (100‐fold) on the identities of base pairs in the aptamer stem suggested that the stem region, which is conserved in length but variable in base‐pair composition and context, is the tuning region of the FMN‐specific aptamer. Synthetic riboswitches were constructed based on the same aptamer domain by rationally modifying the tuning regions. The observed 9.31‐fold difference in the half‐maximal effective concentration (EC50) corresponded to a 11.6‐fold difference in the dissociation constant (KD) of the aptamer domains and suggested that the gene expression can be controlled by rationally adjusting the tuning regions.  相似文献   

5.
6.
In bacteria, the binding between the riboswitch aptamer domain and ligand is regulated by environmental cues, such as low Mg2+ in macrophages during pathogenesis to ensure spatiotemporal expression of virulence genes. Binding was investigated between the flavin mononucleotide (FMN) riboswitch aptamer and its anionic ligand in the presence of molecular crowding agent without Mg2+ ion, which mimics pathogenic conditions. Structural, kinetic, and thermodynamic analyses under the crowding revealed more dynamic conformational rearrangements of the FMN riboswitch aptamer compared to dilute Mg2+‐containing solution. It is hypothesized that under crowding conditions FMN binds through an induced fit mechanism in contrast to the conformational selection mechanism previously demonstrated in dilute Mg2+solution. Since these two mechanisms involve different conformational intermediates and rate constants, these findings have practical significance in areas such as drug design and RNA engineering.  相似文献   

7.
To explore folding and ligand recognition of metabolite-responsive RNAs is of major importance to comprehend gene regulation by riboswitches. Here, we demonstrate, using NMR spectroscopy, that the free aptamer of a preQ(1) class I riboswitch preorganizes into a pseudoknot fold in a temperature- and Mg(2+)-dependent manner. The preformed pseudoknot represents a structure that is close to the ligand-bound state and that likely represents the conformation selected by the ligand. Importantly, a defined base pair mutation within the pseudoknot interaction stipulates whether, in the absence of ligand, dimer formation of the aptamer competes with intramolecular pseudoknot formation. This study pinpoints how RNA preorganization is a crucial determinant for the adaptive recognition process of RNA and ligand.  相似文献   

8.
9.
Riboswitches regulate gene expression via specific recognition of cognate metabolites by their aptamer domains, which fold into stable conformations upon ligand binding. However, the recently reported solution and crystal structures of the Bacillus subtilis preQ(1) riboswitch aptamer show small but significant differences, suggesting that there may be conformational heterogeneity in the ligand-bound state. We present a structural and dynamic characterization of this aptamer by solution NMR spectroscopy. The aptamer-preQ(1) complex is intrinsically flexible in solution, with two regions that undergo motions on different time scales. Three residues move in concert on the micro-to-millisecond time scale and may serve as the lid of the preQ(1)-binding pocket. Several Ca(2+) ions are present in the crystal structure, one of which binds with an affinity of 47 ± 2 μM in solution to a site that is formed only upon ligand binding. Addition of Ca(2+) to the aptamer-preQ(1) complex in solution results in conformational changes that account for the differences between the solution and crystal structures. Remarkably, the Ca(2+) ions present in the crystal structure, which were proposed to be important for folding and ligand recognition, are not required for either in solution.  相似文献   

10.
Conformational dynamics and observed equilibrium constants for ligand binding of the adenine-specific riboswitch (add-A riboswitch) in the absence of Mg(2+) and presence of various concentrations of poly-ethylene glycol having an average molecular weight of 200 indicated that 54.2 water molecules were released from P2 and P3 stem regions of the add-A riboswitch during conformational transition upon the binding of 2-aminopurine, an analog of the natural ligand adenine.  相似文献   

11.
12.
13.
BACKGROUND: Anti-idiotype approaches are based on the assumption that an antibody recognising a ligand can be structurally related to the receptor. Recently we have generated anti-idiotype RNA aptamers designed to mimic the human immunodeficiency virus-1 (HIV-1) Rev nuclear export signal (NES). Nuclear injection of either NES-peptide conjugates or aptamer causes the inhibition of Rev-mediated export. This implied that NES mimics and export substrate might compete for binding to the NES receptor. The mechanism of inhibition, however, is unknown. RESULTS: The interaction between the export aptamer and CRM1 was characterised in vitro. The aptamer binds specifically to CRM1 and this interaction is sensitive to competition by Rev NES-peptide conjugates. The recognition domain of CRM1 has been mapped and includes residues found previously to affect binding of leptomycin B, a fungicide interfering with nuclear export. CONCLUSIONS: Inhibition of Rev-mediated export in vivo by export aptamers appears to result from the binding of the aptamers to the NES-recognition domain of CRM1. This observation demonstrates that anti-idiotype RNA can mimic faithfully structural and functional properties of a protein and can be used to map ligand-binding domains of receptors.  相似文献   

14.
We have investigated the effect of the folding of DNA aptamers on the colloidal stability of gold nanoparticles (AuNPs) to which an aptamer is tethered. On the basis of the studies of two different aptamers (adenosine aptamer and K+ aptamer), we discovered a unique colloidal stabilization effect associated with aptamer folding: AuNPs to which folded aptamer structures are attached are more stable toward salt-induced aggregation than those tethered to unfolded aptamers. This colloidal stabilization effect is more significant when a DNA spacer was incorporated between AuNP and the aptamer or when lower aptamer surface graft densities were used. The conformation that aptamers adopt on the surface appears to be a key factor that determines the relative stability of different AuNPs. Dynamic light scattering experiments revealed that the sizes of AuNPs modified with folded aptamers were larger than those of AuNPs modified with unfolded (but largely collapsed) aptamers in salt solution. From both the electrostatic and steric stabilization points of view, the folded aptamers that are more extended from the surface have a higher stabilization effect on AuNP than the unfolded aptamers. On the basis of this unique phenomenon, colorimetric biosensors have been developed for the detection of adenosine, K+, adenosine deaminase, and its inhibitors. Moreover, distinct AuNP aggregation and redispersion stages can be readily operated by controlling aptamer folding and unfolding states with the addition of adenosine and adenosine deaminase.  相似文献   

15.
The glycine riboswitch regulates gene expression through the cooperative recognition of its amino acid ligand by a tandem pair of aptamers. A 3.6 ? crystal structure of the tandem riboswitch from the glycine permease operon of Fusobacterium nucleatum reveals the glycine binding sites and an extensive network of interactions, largely mediated by asymmetric A-minor contacts, that serve to communicate ligand binding status between the aptamers. These interactions provide a structural basis for how the glycine riboswitch cooperatively regulates gene expression.  相似文献   

16.
The 68 residue peptide IA 3 is an intrinsically unstructured protein that serves as an endogenous inhibitor of the yeast aspartic proteinase A (YPrA). Although unstructured in free solution, IA 3 forms an N-terminal alpha helix as it binds to YPrA, leading to subnanomolar inhibition of the protease. Equilibrium structural and inhibition studies provide little insight into the mechanism and kinetics of the coupled folding and binding interaction. We have used laser temperature jump spectroscopy to study the kinetics of folding of free IA 3 and of the interaction between IA 3 and YPrA. Inducing folding with trifluoroethanol cosolvent allows us to determine the folding rate (kf approximately 0.3 (micros)(-1)) and the unfolding rate (ku approximately 3 (micros)(-1)) for free IA 3 in water at 25 degrees C. A substantially faster relaxation process is observed in the presence of the proteinase; this process appears to be the kinetic signature of an intermediate binding step in the coupled folding and binding interaction of IA 3 and YPrA.  相似文献   

17.
Reductive unfolding studies of proteins are designed to provide information about intramolecular interactions that govern the formation (and stabilization) of the native state and about folding/unfolding pathways. By mutating Tyr92 to G, A, or L in the model protein, bovine pancreatic ribonuclease A, and through analysis of temperature factors and molecular dynamics simulations of the crystal structures of these mutants, it is demonstrated that the markedly different reductive unfolding rates and pathways of ribonuclease A and its structural homologue onconase can be attributed to a single, localized, ring-stacking interaction between Tyr92 and Pro93 in the bovine variant. The fortuitous location of this specific stabilizing interaction in a disulfide-bond-containing loop region of ribonuclease A results in the localized modulation of protein dynamics that, in turn, enhances the susceptibility of the disulfide bond to reduction leading to an alteration in the reductive unfolding behavior of the homologues. These results have important implications for folding studies involving topological determinants to obtain folding/unfolding rates and pathways, for protein structure-function prediction through fold recognition, and for predicting proteolytic cleavage sites.  相似文献   

18.
Molecular recognition of small molecule ligands by the nucleic acid aptamers for tobramycin, ATP, and FMN has been examined using electrospray ionization mass spectrometry (ESI-MS). Mass spectrometric data for binding stoichiometry and relative binding affinity correlated well with solution data for tobramycin aptamer complexes, in which aptamer/ligand interactions are mediated by hydrogen bonds. For the ATP and FMN aptamers, where ligand interactions involve both hydrogen bonding and significant pi-stacking, the relative binding affinities determined by MS did not fully correlate with results obtained from solution experiments. Some high-affinity aptamer/ligand complexes appeared to be destabilized in the gas phase by internal Coulombic repulsion. In CAD experiments, complexes with a greater number of intermolecular hydrogen bonds exhibited greater gas-phase stability even in cases when solution binding affinities were equivalent. These results indicate that in at least some cases, mass spectrometric data on aptamer/ligand binding affinities should be used in conjunction with complementary techniques to fully assess aptamer molecular recognition properties.  相似文献   

19.
Probing the structure of DNA aptamers with a classic heterocycle   总被引:1,自引:0,他引:1  
DNA aptamers are synthetic, single-stranded DNA oligonucleotides selected by SELEX methods for their binding with specific ligands. Here we present ethidium binding results for three related DNA aptamers (PDB code: 1OLD, 1DB6, and 2ARG)that bind L-argininamide (L-Arm). The ligand bound form of each aptamer's structure has been reported and each are found to be composed primarily of two domains consisting of a stem helical region and a loop domain that forms a binding pocket for the cognate ligand. Previous thermodynamic experiments demonstrated that the DNA aptamer 1OLD undergoes a large conformational ordering upon binding to L-Arm. Here we extend those linkage binding studies by examining the binding of the heterocyclic intercalator ethidium to each of the three aptamers by fluorescence and absorption spectrophotometric titrations. Our results reveal that ethidium binds to each aptamer with DeltaG degree's in the range of -8.7 to -9.4 kcal/mol. The stoichiometry of binding is 2:1 for each aptamer and is quantitatively diminished in the presence of L-Arm as is the overall fluorescence intensity of ethidium. Together, these results demonstrate that a portion of the bound ethidium is excluded from the aptamer in the presence of a saturating amount of L-Arm. These results demonstrate the utility of ethidium and related compounds for the probing of non-conventional DNA structures and reveal an interesting fundamental thermodynamic linkage in DNA aptamers. Results are discussed in the context of the thermodynamic stability and structure of each of the aptamers examined.  相似文献   

20.
Intrinsically disordered proteins (IDPs) play crucial roles in protein interaction networks and in this context frequently constitute important hubs and interfaces. Here we show by a combination of NMR and EPR spectroscopy that the binding of the cytokine osteopontin (OPN) to its natural ligand, heparin, is accompanied by thermodynamically compensating structural adaptations. The core segment of OPN expands upon binding. This “unfolding‐upon‐binding” is governed primarily through electrostatic interactions between heparin and charged patches along the protein backbone and compensates for entropic penalties due to heparin–OPN binding. It is shown how structural unfolding compensates for entropic losses through ligand binding in IDPs and elucidates the interplay between structure and thermodynamics of rapid substrate‐binding and ‐release events in IDP interaction networks.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号