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.
Many RNA structures are composed of simple secondary structure elements linked by a few critical tertiary interactions. SHAPE chemistry has made interrogation of RNA dynamics at single-nucleotide resolution straightforward. However, de novo identification of nucleotides involved in tertiary interactions remains a challenge. Here we show that nucleotides that form noncanonical or tertiary contacts can be detected by comparing information obtained using two SHAPE reagents, N-methylisatoic anhydride (NMIA) and 1-methyl-6-nitroisatoic anhydride (1M6). Nucleotides that react preferentially with NMIA exhibit slow local nucleotide dynamics and usually adopt the less common C2'-endo ribose conformation. Experiments and first-principles calculations show that 1M6 reacts preferentially with nucleotides in which one face of the nucleobase allows an unhindered stacking interaction with the reagent. Differential SHAPE reactivities were used to detect noncanonical and tertiary interactions in four RNAs with diverse structures and to identify preformed noncanonical interactions in partially folded RNAs. Differential SHAPE reactivity analysis will enable experimentally concise, large-scale identification of tertiary structure elements and ligand binding sites in complex RNAs and in diverse biological environments.  相似文献   

2.
The functions of most RNA molecules are critically dependent on the distinct local dynamics that characterize secondary structure and tertiary interactions and on structural changes that occur upon binding by proteins and small molecule ligands. Measurements of RNA dynamics at nucleotide resolution set the foundation for understanding the roles of individual residues in folding, catalysis, and ligand recognition. In favorable cases, local order in small RNAs can be quantitatively analyzed by NMR in terms of a generalized order parameter, S2. Alternatively, SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) chemistry measures local nucleotide flexibility in RNAs of any size using structure-sensitive reagents that acylate the 2'-hydroxyl position. In this work, we compare per-residue RNA dynamics, analyzed by both S2 and SHAPE, for three RNAs: the HIV-1 TAR element, the U1A protein binding site, and the Tetrahymena telomerase stem loop 4. We find a very strong correlation between the two measurements: nucleotides with high SHAPE reactivities consistently have low S2 values. We conclude that SHAPE chemistry quantitatively reports local nucleotide dynamics and can be used with confidence to analyze dynamics in large RNAs, RNA-protein complexes, and RNAs in vivo.  相似文献   

3.
Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry yields quantitative RNA secondary and tertiary structure information at single nucleotide resolution. SHAPE takes advantage of the discovery that the nucleophilic reactivity of the ribose 2'-hydroxyl group is modulated by local nucleotide flexibility in the RNA backbone. Flexible nucleotides are reactive toward hydroxyl-selective electrophiles, whereas constrained nucleotides are unreactive. Initial versions of SHAPE chemistry, which employ isatoic anhydride derivatives that react on the minute time scale, are emerging as the ideal technology for monitoring equilibrium structures of RNA in a wide variety of biological environments. Here, we extend SHAPE chemistry to a benzoyl cyanide scaffold to make possible facile time-resolved kinetic studies of RNA in approximately 1 s snapshots. We then use SHAPE chemistry to follow the time-dependent folding of an RNase P specificity domain RNA. Tertiary interactions form in two distinct steps with local tertiary contacts forming an order of magnitude faster than long-range interactions. Rate-determining tertiary folding requires minutes despite that no non-native interactions must be disrupted to form the native structure. Instead, overall folding is limited by simultaneous formation of interactions approximately 55 A distant in the RNA. Time-resolved SHAPE holds broad potential for understanding structural biogenesis and the conformational interconversions essential to the functions of complex RNA molecules at single nucleotide resolution.  相似文献   

4.
The biological functions of RNA are ultimately governed by the local environment at each nucleotide. Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry is a powerful approach for measuring nucleotide structure and dynamics in diverse biological environments. SHAPE reagents acylate the 2'-hydroxyl group at flexible nucleotides because unconstrained nucleotides preferentially sample rare conformations that enhance the nucleophilicity of the 2'-hydroxyl. The critical corollary is that some constrained nucleotides must be poised for efficient reaction at the 2'-hydroxyl group. To identify such nucleotides, we performed SHAPE on intact crystals of the Escherichia coli ribosome, monitored the reactivity of 1490 nucleotides in 16S rRNA, and examined those nucleotides that were hyper-reactive toward SHAPE and had well-defined crystallographic conformations. Analysis of these conformations revealed that 2'-hydroxyl reactivity is broadly facilitated by general base catalysis involving multiple RNA functional groups and by two specific orientations of the bridging 3'-phosphate group. Nucleotide analog studies confirmed the contributions of these mechanisms to SHAPE reactivity. These results provide a strong mechanistic explanation for the relationship between SHAPE reactivity and local RNA dynamics and will facilitate interpretation of SHAPE information in the many technologies that make use of this chemistry.  相似文献   

5.
A precise tertiary structure must be adopted to allow the function of many RNAs in cells. Accordingly, increasing resources have been devoted to the elucidation of RNA structures and the folding of RNAs. 2-Aminopurine (2AP), a fluorescent nucleobase analogue, can be substituted in strategic positions of DNA or RNA molecules to act as site-specific probe to monitor folding and folding dynamics of nucleic acids. Recent studies further demonstrated the potential of 2AP modifications in the assessment of folding kinetics during ligand-induced secondary and tertiary RNA structure rearrangements. However, an efficient way to unambiguously identify reliable positions for 2AP sensors is as yet unavailable and would represent a major asset, especially in the absence of crystallographic or NMR structural data for a target molecule. We report evidence of a novel and direct correlation between the 2'-OH flexibility of nucleotides, observed by selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) probing and the fluorescence response following nucleotide substitutions by 2AP. This correlation leads to a straightforward method, using SHAPE probing with benzoyl cyanide, to select appropriate nucleotide sites for 2AP substitution. This clear correlation is presented for three model RNAs of biological significance: the SAM-II, adenine (addA), and preQ(1) class II (preQ(1)cII) riboswitches.  相似文献   

6.
7.
Template-directed polymerization of chemically activated ribonucleotide monomers, such as nucleotide 5'-phosphorimidazolides, has been studied as a model for nonenzymatic RNA replication during the origin of life. Kinetic studies of the polymerization of various nucleotide monomers on oligonucleotide templates have suggested that the A-form (C3'-endo sugar pucker) conformation is optimal for both monomers and templates for efficient copying. However, RNA monomers are predominantly in the C2'-endo conformation when free in solution, except for cytidine, which is approximately equally distributed between the C2'-endo and C3'-endo conformations. We hypothesized that ribonucleotides undergo a switch in sugar pucker upon binding to an A-type template and that this conformational switch allows or enhances subsequent polymerization. We used transferred nuclear Overhauser effect spectroscopy (TrNOESY), which can be used for specific detection of the bound conformation of small-molecule ligands with relatively weak affinity to receptors, to study the interactions between nucleotide 5'-phosphorimidazolides and single-stranded oligonucleotide templates. We found that the sugar pucker of activated ribonucleotides switches from C2'-endo in the free state to C3'-endo upon binding to an RNA template. This switch occurs only on RNA and not on DNA templates. Furthermore, activated 2'-deoxyribonucleotides maintain a C2'-endo sugar pucker in both the free and template-bound states. Our results provide a structural explanation for the observations that activated ribonucleotides are superior to activated deoxyribonucleotides and that RNA templates are superior to DNA templates in template-directed nonenzymatic primer-extension reactions.  相似文献   

8.
9.
In vitro selection was used to identify deoxyribozymes that ligate two RNA substrates. In the ligation reaction, a 2'-5' RNA phosphodiester linkage is created from a 2',3'-cyclic phosphate and a 5'-hydroxyl group. The new Mg(2+)-dependent deoxyribozymes provide 50-60% yield of ligated RNA in overnight incubations at pH 7.5 and 37 degrees C, and they afford 40-50% yield in 1 h at pH 9.0 and 37 degrees C. Various RNA substrate sequences may be joined by simple Watson-Crick covaration of the DNA binding arms that interact with the two RNA substrates. The current deoxyribozymes have some RNA substrate sequence requirements at the nucleotides immediately surrounding the ligation junction (either UAUA GGAA or UAUN GGAA, where the arrow denotes the ligation site and N equals any nucleotide). One of the new deoxyribozymes was used to prepare by ligation the Tetrahymena group I intron RNA P4-P6 domain, a representative structured RNA. Nondenaturing gel electrophoresis revealed that a 2'-5' linkage between nucleotides A233 and G234 of P4-P6 does not disrupt its Mg(2+)-dependent folding (DeltaDeltaG degrees ' < 0.2 kcal/mol). This demonstrates that a 2'-5' linkage does not necessarily interfere with structure in a folded RNA. Therefore, these non-native linkages may be acceptable in modified RNAs when structure/function relationships are investigated. Deoxyribozymes that ligate RNA should be particularly useful for preparing site-specifically modified RNAs for studies of RNA structure, folding, and catalysis.  相似文献   

10.
11.
G-rich nucleic acid oligomers can form G-quadruplexes built by G-tetrads stacked upon each other. Depending on the nucleotide sequence, G-quadruplexes fold mainly with two topologies: parallel, in which all G-tracts are oriented parallel to each other, or antiparallel, in which one or more G-tracts are oriented antiparallel to the other G-tracts. In the former topology, all glycosidic bond angles conform to anti conformations, while in the latter topology they adopt both syn and anti conformations. It is of interest to understand the molecular forces that govern G-quadruplex folding. Here, we approach this problem by examining the impact of LNA (locked nucleic acid) modifications on the folding topology of the dimeric model system of the human telomere sequence. In solution, this DNA G-quadruplex forms a mixture of G-quadruplexes with antiparallel and parallel topologies. Using CD and NMR spectroscopies, we show that LNA incorporations can modulate this equilibrium in a rational manner and we establish a relationship between incorporation of LNA nucleotides in syn and/or anti positions and the shift of the equilibrium to obtain exclusively the parallel G-quadruplex. The change in topology is driven by a combination of the C3'-endo puckering of LNA nucleotides and their preference for the anti glycosidic conformation. In addition, the parallel LNA-modified G-quadruplexes are thermally stabilised by about 11 °C relative to their DNA counterparts.  相似文献   

12.
13.
14.
15.
Ribose 2'-amine substitutions are broadly useful as structural probes in nucleic acids. In addition, structure-selective chemical reaction at 2'-amine groups is a robust technology for interrogating local nucleotide flexibility and conformational changes in RNA and DNA. We analyzed crystal structures for several RNA duplexes containing 2'-amino cytidine (C(N)) residues that form either C(N)-G base pairs or C(N)-A mismatches. The 2'-amine substitution is readily accommodated in an A-form RNA helix and thus differs from the C2'-endo conformation observed for free nucleosides. The 2'-amide product structure was visualized directly by acylating a C(N)-A mismatch in intact crystals and is also compatible with A-form geometry. To visualize conformations able to facilitate formation of the amide-forming transition state, in which the amine nucleophile carries a positive partial charge, we analyzed crystals of the C(N)-A duplex at pH 5, where the 2'-amine is protonated. The protonated amine moves to form a strong electrostatic interaction with the 3'-phosphodiester. Taken together with solution-phase experiments, 2'-amine acylation is likely facilitated by either of two transition states, both involving precise positioning of the adjacent 3'-phosphodiester group.  相似文献   

16.
The RNA helicase DbpA promotes RNA remodeling coupled to ATP hydrolysis. It is unique because of its specificity to hairpin 92 of 23S rRNA (HP92). Although DbpA kinetic pathways leading to ATP hydrolysis and RNA unwinding have been recently elucidated, the molecular (atomic) basis for the coupling of ATP hydrolysis to RNA remodeling remains unclear. This is, in part, due to the lack of detailed structural information on the ATPase site in the presence and absence of RNA in solution. We used high-field pulse ENDOR (electron-nuclear double resonance) spectroscopy to detect and analyze fine conformational changes in the protein's ATPase site in solution. Specifically, we substituted the essential Mg(2+) cofactor in the ATPase active site for paramagnetic Mn(2+) and determined its close environment with different nucleotides (ADP, ATP, and the ATP analogues ATPγS and AMPPnP) in complex with single- and double-stranded RNA. We monitored the Mn(2+) interactions with the nucleotide phosphates through the (31)P hyperfine couplings and the coordination by protein residues through (13)C hyperfine coupling from (13)C-enriched DbpA. We observed that the nucleotide binding site of DbpA adopts different conformational states upon binding of different nucleotides. The ENDOR spectra revealed a clear distinction between hydrolyzable and nonhydrolyzable nucleotides prior to RNA binding. Furthermore, both the (13)C and the (31)P ENDOR spectra were found to be highly sensitive to changes in the local environment of the Mn(2+) ion induced by the hydrolysis. More specifically, ATPγS was efficiently hydrolyzed upon binding of RNA, similar to ATP. Importantly, the Mn(2+) cofactor remains bound to a single protein side chain and to one or two nucleotide phosphates in all complexes, whereas the remaining metal coordination positions are occupied by water. The conformational changes in the protein's ATPase active site associated with the different DbpA states occur in remote coordination shells of the Mn(2+) ion. Finally, a competitive Mn(2+) binding site was found for single-stranded RNA construct.  相似文献   

17.
RNA folding dynamics plays important roles in various functions of RNAs. To date, coarse-grained modeling has been successfully employed to simulate RNA folding dynamics on the energy landscape composed of secondary structures. In such a modeling, the energy barrier height between metastable structures is a key parameter that crucially affects the simulation results. Although a number of approaches ranging from the exact method to heuristic ones are available to predict the barrier heights, developing an efficient heuristic for this purpose is still an algorithmic challenge.We developed a novel RNA folding pathway prediction method, ACOfoldpath, based on Ant Colony Optimization (ACO). ACO is a widely used powerful combinatorial optimization algorithm inspired from the food-seeking behavior of ants. In ACOfoldpath, to accelerate the folding pathway prediction, we reduce the search space by utilizing originally devised structure generation rules. To evaluate the performance of the proposed method, we benchmarked ACOfoldpath on the known nineteen conformational RNA switches. As a result, ACOfoldpath successfully predicted folding pathways better than or comparable to the previous heuristics. The results of RNA folding dynamics simulations and pseudoknotted pathway predictions are also presented.  相似文献   

18.
Arylazide mediated photocrosslinking has been widely used to obtain structural constraints in biological systems, even though the reactive species generated upon photolysis in aqueous solution have not been well characterized. We establish a mechanistic framework for formation of adducts between photoactivated 3-hydroxyphenyl azide and RNA. Tethered to an internal site in an RNA duplex via a 2'-amido linkage, photolysis of the aryl azide yields a cross-strand cross-link. Analysis of the ability of reagents with diagnostic reactivities to intercept formation of this cross-strand cross-link supports the assignment that the photoactivated intermediate is the ketenimine or a ketenimine-derived ring expansion product. Neither the initially produced singlet nitrene nor the subsequently formed triplet nitrene contribute to cross-link formation. Argon matrix and time-resolved solution experiments show that photolysis of free 3-hydroxyphenyl azide releases (in 相似文献   

19.
20.
MD simulations of homomorphous single-stranded PNA, DNA, and RNA with the same base sequence have been performed in aqueous solvent. For each strand two separate simulations were performed starting from a (i) helical conformation and (ii) random coiled state. Comparisons of the simulations with the single-stranded helices (case i) show that the differences in the covalent nature of the backbones cause significant differences in the structural and dynamical properties of the strands. It is found that the PNA strand maintains its nice base-stacked initial helical structure throughout the 1.5-ns MD simulation at 300 K, while DNA/RNA show relatively larger fluctuations in the structures with a few local unstacking events during -ns MD simulation each. It seems that the weak physical coupling between the bases and the backbone in PNA causes a loss of correlation between the dynamics of the bases and the backbone compared to the DNA/RNA and helps maintain the base-stacked helical conformation. The global flexibility of a single-stranded PNA helix was also found to be lowest, while RNA appears to be the most flexible single-stranded helix. The sugar pucker of several nucleotides in single-stranded DNA and RNA was found to adopt both C2'-endo and C3'-endo conformations for significant times. This effect is more pronounced for single strands in completely coiled states. The simulations with single-stranded coils as the initial structure also indicate that a PNA can adopt a more compact globular structure, while DNA/RNA of the same size adopts a more extended coil structure. This allows even a short PNA in the coiled state to form a significantly stable nonsequentially base-stacked globular structure in solution. Due to the hydrophobic nature of the PNA backbone, it interacts with surrounding water rather weakly compared to DNA/RNA.  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