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 62 毫秒
1.
Diamond JM  Turner DH  Mathews DH 《Biochemistry》2001,40(23):6971-6981
RNA multibranch loops (junctions) are loops from which three or more helices exit. They are nearly ubiquitous in RNA secondary structures determined by comparative sequence analysis. In this study, systems in which two strands combine to form three-way junctions were used to measure the stabilities of RNA multibranch loops by UV optical melting and isothermal titration calorimetry (ITC). These data were used to calculate the free energy increment for initiation of a three-way junction on the basis of a nearest neighbor model for secondary structure stability. Imino proton NMR spectra were also measured for two systems and are consistent with the hypothesized helical structures. Incorporation of the experimental data into the mfold and RNA structure computer programs has contributed to an improvement in prediction of RNA secondary structure from sequence.  相似文献   

2.
Three-way multibranch loops (junctions) are common in RNA secondary structures. Computer algorithms such as RNAstructure and MFOLD do not consider the identity of unpaired nucleotides in multibranch loops when predicting secondary structure. There is limited experimental data, however, to parametrize this aspect of these algorithms. In this study, UV optical melting and a fluorescence competition assay are used to measure stabilities of multibranch loops containing up to five unpaired adenosines or uridines or a loop E motif. These results provide a test of our understanding of the factors affecting multibranch loop stability and provide revised parameters for predicting stability. The results should help to improve predictions of RNA secondary structure.  相似文献   

3.
Competition binding and UV melting studies of a DNA model system consisting of three, four or five mutually complementary oligonucleotides demonstrate that unpaired bases at the branch point stabilize three- and five-way junction loops but destabilize four-way junctions. The inclusion of unpaired nucleotides permits the assembly of five-way DNA junction complexes (5WJ) having as few as seven basepairs per arm from five mutually complementary oligonucleotides. Previous work showed that 5WJ, having eight basepairs per arm but lacking unpaired bases, could not be assembled [Wang, Y.L., Mueller, J.E., Kemper, B. and Seeman, N.C. (1991) Biochemistry, 30, 5667-5674]. Competition binding experiments demonstrate that four-way junctions (4WJ) are more stable than three-way junctions (3WJ), when no unpaired bases are included at the branch point, but less stable when unpaired bases are present at the junction. 5WJ complexes are in all cases less stable than 4WJ or 3WJ complexes. UV melting curves confirm the relative stabilities of these junctions. These results provide qualitative guidelines for improving the way in which multi-helix junction loops are handled in secondary structure prediction programs, especially for single-stranded nucleic acids having primary sequences that can form alternative structures comprising different types of junctions.  相似文献   

4.
Klostermeier D  Millar DP 《Biochemistry》2000,39(42):12970-12978
Helical junctions are ubiquitous structural elements that govern the folding and tertiary structure of RNAs. The tobacco ringspot virus hairpin ribozyme consists of two helix-loop-helix elements that lie on adjacent arms of a four-way junction. In the active form of the hairpin ribozyme, the loops are in proximity. The nature of the helical junction determines the stability of the hairpin ribozyme tertiary structure [Walter, N. G., Burke, J. M., and Millar, D. P. (1999) Nat. Struct. Biol. 6, 544-549] and thus its catalytic activity. We used two-, three-, and four-way junction hairpin ribozymes as model systems to investigate the thermodynamic basis for the different tertiary structure stabilities. The equilibrium between docked and extended conformers was analyzed as a function of temperature using time-resolved fluorescence resonance energy transfer (trFRET). As the secondary and tertiary structure transitions overlap, information from UV melting curves and trFRET had to be combined to gain insight into the thermodynamics of both structural transitions. It turned out that the higher tertiary structure stability observed in the context of a four-way junction is the result of a lower entropic cost for the docking process. In the two- and three-way junction ribozymes, a high entropic cost counteracts the favorable enthalpic term, rendering the docked conformer only marginally stable. Thus, two- and three-way junction tertiary structures are more sensitive toward regulation by ligands, whereas four-way junctions provide a stable scaffold. Altogether, RNA folding and stability appear to be governed by principles similar to those for the folding of proteins.  相似文献   

5.
We have studied the structure of a number of three-way DNA junctions that were closely related in sequence to four-way junctions studied previously. We observe that the electrophoretic mobility of the species derived by selective shortening of one arm of a junction are very similar whichever arm is shortened, and that this remains so whether or not magnesium is present in the buffer. This suggests that the angles subtended between the arms of the three-way junctions are similar. All thymine bases located immediately at the junction are reactive to osmium tetroxide, indicating that out-of-plane attack is not prevented by helix-helix stacking, and this is also independent of the presence or absence of metal cations. The results suggest that the three-way junction cannot undergo an ion-induced conformational folding involving helical stacking, but remains fixed in a Y-shaped extended conformation. Thus the three- and four-way junctions are quite different in character in the presence of cations.  相似文献   

6.
In the present paper, the interactions of the origin binding protein (OBP) of herpes simplex virus type 1 (HSV1) with synthetic four-way Holliday junctions (HJs) were studied using electrophoresis mobility shift assay and the FRET method and compared with the interactions of the protein with duplex and single-stranded DNAs. It has been found that OBP exhibits a strong preference for binding to four-way and three-way DNA junctions and possesses much lower affinities to duplex and single-stranded DNAs. The protein forms three types of complexes with HJs. It forms complexes I and II which are reminiscent of the tetramer and octamer complexes with four-way junction of HJ-specific protein RuvA of Escherichia coli. The binding approaches saturation level when two OBP dimers are bound per junction. In the presence of Mg2+ ions (≥2 mM) OBP also interacts with HJ in the stacked arm form (complex III). In the presence of 5 mM ATP and 10 mM Mg2+ ions OBP catalyzes processing of the HJ in which one of the annealed oligonucleotides has a 3′-terminal tail containing 20 unpaired thymine residues. The observed preference of OBP for binding to the four-way DNA junctions provides a basis for suggestion that OBP induces large DNA structural changes upon binding to Box I and Box II sites in OriS. These changes involve the bending and partial melting of the DNA at A+T-rich spacer and also include the formation of HJ containing Box I and Box II inverted repeats and flanking DNA sequences.  相似文献   

7.
Lilley DM 《Biopolymers》1998,48(2-3):101-112
The global structures of branched RNA species are important to their function. Branched RNA species are defined as molecules in which double-helical segments are interrupted by abrupt discontinuities. These include helical junctions of different orders, and base bulges and loops. Common helical junctions are three- and four-way junctions, often interrupted by mispairs or additional nucleotides. There are many interesting examples of functional RNA junctions, including the hammerhead and hairpin ribozymes, and junctions that serve as binding sites for proteins. The junctions display some common structural properties. These include a tendency to undergo pairwise helical stacking and ion-induced conformational transitions. Helical branchpoints can act as key architectural components and as important sites for interactions with proteins. Copyright 1999 John Wiley & Sons, Inc.  相似文献   

8.
RNA junctions are important structural elements that form when three or more helices come together in space in the tertiary structures of RNA molecules. Determining their structural configuration is important for predicting RNA 3D structure. We introduce a computational method to predict, at the secondary structure level, the coaxial helical stacking arrangement in junctions, as well as classify the junction topology. Our approach uses a data mining approach known as random forests, which relies on a set of decision trees trained using length, sequence and other variables specified for any given junction. The resulting protocol predicts coaxial stacking within three- and four-way junctions with an accuracy of 81% and 77%, respectively; the accuracy increases to 83% and 87%, respectively, when knowledge from the junction family type is included. Coaxial stacking predictions for the five to ten-way junctions are less accurate (60%) due to sparse data available for training. Additionally, our application predicts the junction family with an accuracy of 85% for three-way junctions and 74% for four-way junctions. Comparisons with other methods, as well applications to unsolved RNAs, are also presented. The web server Junction-Explorer to predict junction topologies is freely available at: http://bioinformatics.njit.edu/junction.  相似文献   

9.
F Jensch  H Kosak  N C Seeman    B Kemper 《The EMBO journal》1989,8(13):4325-4334
We have purified a cruciform DNA resolving endonuclease (Endo X3) greater than 1000-fold from crude extracts of mitotically growing Saccharomyces cerevisiae. The enzyme shows high specificity for DNAs with secondary structures and introduces characteristic patterns of staggered 'nicks' in the immediate vicinity of the structure. The following substrates were analyzed in detail: (i) naturally occurring four-way X junctions in cruciform DNA of a supercoiled plasmid; (ii) synthetic four-way X junctions with arms of 9 bp; (iii) synthetic three-way Y junctions with arms of 10 bp; and (iv) heteroduplex loops with 19 nucleotides in the loop. Cleavages were always found in the double stranded portion of the DNA, located immediately adjacent to the junction of the respective structure. The Endo X3 induced cleavage patterns are identical or very similar to the cleavage patterns induced in the same substrates by endonuclease VII (Endo VII) from phage T4. Furthermore, the activity of Endo X3 is completely inhibited in the presence of anti-Endo VII antiserum. Endo X3 has an apparent mol. wt of 43,000 daltons, determined by gel filtration and of approximately 18,000 daltons in SDS--polyacrylamide gels. Maximum activity of the enzyme was obtained in the presence of 10 mM MgCl2 at 31 degrees C in Tris-HCl buffer over a broad pH range with a maximum approximately 8.0. About 70% of maximal activity was obtained when Mg2+ was replaced by equimolar amounts of Mn2+ or Ca2+.  相似文献   

10.
The various motifs of RNA molecules are closely related to their structural and functional properties. To better understand the nature and distributions of such structural motifs (i.e., paired and unpaired bases in stems, junctions, hairpin loops, bulges, and internal loops) and uncover characteristic features, we analyze the large 16S and 23S ribosomal RNAs of Escherichia coli. We find that the paired and unpaired bases in structural motifs have characteristic distribution shapes and ranges; for example, the frequency distribution of paired bases in stems declines linearly with the number of bases, whereas that for unpaired bases in junctions has a pronounced peak. Significantly, our survey reveals that the ratio of total (over the entire molecule) unpaired to paired bases (0.75) and the fraction of bases in stems (0.6), junctions (0.16), hairpin loops (0.12), and bulges/internal loops (0.12) are shared by 16S and 23S ribosomal RNAs, suggesting that natural RNAs may maintain certain proportions of bases in various motifs to ensure structural integrity. These findings may help in the design of novel RNAs and in the search (via constraints) for RNA-coding motifs in genomes, problems of intense current focus.  相似文献   

11.
Non-paired nucleotides stabilize the formation of three-way helical DNA junctions. Two or more unpaired nucleotides located in the junction region enable oligomers ten to fifteen nucleotides long to assemble, forming conformationally homogeneous junctions, as judged by native gel electrophoresis. The unpaired bases can be present on the same strand or on two different strands. Up to five extra bases on one strand have been tested and found to produce stable junctions. The formation of stable structures is favored by the presence of a divalent cation such as magnesium and by high monovalent salt concentration. The order-disorder transition of representative three-way junctions was monitored optically in the ultraviolet and analyzed to quantify thermodynamically the stabilization provided by unpaired bases in the junction region. We report the first measurements of the thermodynamics of adding an unpaired nucleotide to a nucleic acid three-way junction. We find that delta delta G degrees (37 degrees C) = +0.5 kcal/mol for increasing the number of unpaired adenosines from two to three. Three-way junctions having reporter arms 40 base-pairs long were also prepared. Each of the three reporter arms contained a unique restriction site 15 base-pairs from the junction. Asymmetric complexes produced by selectively cleaving each arm were analyzed on native gels. Cleavage of the double helical arm opposite the strand having the two extra adenosines resulted in a complex that migrated more slowly than complexes produced by cleavage at either of the other two arms. It is likely that the strand containing the unpaired adenosines is kinked at an acute angle, forming a Y-shaped, rather than a T-shaped junction.  相似文献   

12.
Our knowledge of the architectural principles of nucleic acid junctions has seen significant recent advances. The conformation of DNA junctions is now well understood, and this provides a new basis for the analysis of important structural elements in RNA. The most significant new data have come from X-ray crystallography of four-way DNA junctions; incidentally showing the great importance of serendipity in science, since none of the three groups had deliberately set out to crystallise a junction. Fortunately the results confirm, and of course extend, the earlier conformational studies of DNA junctions in almost every detail. This is important, because it means that these methods can be applied with greater confidence to new systems, especially in RNA. Methods like FRET, chemical probing and even the humble polyacrylamide gel can be rapid and very powerful, allowing the examination of a large number of sequence variants relatively quickly. Molecular modelling in conjunction with experiments is also a very important component of the general approach. Ultimately crystallography provides the gold standard for structural analysis, but the other, simple approaches have considerable value along the way. At the beginning of this review I suggested two simple folding principles for branched nucleic acids, and it is instructive to review these in the light of recent data. In brief, these were the tendency for pairwise coaxial stacking of helical arms, and the importance of metal ion interactions in the induction of folding. We see that both are important in a wide range of systems, both in DNA and RNA. The premier example is the four-way DNA junction, which undergoes metal ion-induced folding into the stacked X-structure that is based on coaxial stacking of arms. As in many systems, there are two alternative ways to achieve this depending on the choice of stacking partners. Recent data reveal that both forms often exist in a dynamic equilibrium, and that the relative stability of the two conformers depends upon base sequence extending a significant distance from the junction. The three-way junction has provided a good test of the folding principles. Perfect three-way (3H) DNA junctions seem to defy these principles in that they appear reluctant to undergo coaxial stacking of arms, and exhibit little change in conformation with addition of metal ions. Modelling suggests that such a junction is stereochemically constrained in an extended conformation. However, upon inclusion of a few additional base pairs at the centre (to create a 3HS2 junction for example) the additional stereochemical flexibility allows two arms to undergo coaxial stacking. Such a junction exhibits all the properties consistent with the general folding principles, with ion-induced folding into a form based on pairwise coaxial stacking of arms in one of two different conformers. The three-way junction is therefore very much the exception that proves the rule. It is instructive to compare the folding of corresponding species in DNA and RNA, where we find both similarities and differences. The RNA four-way junction can adopt a structure that is globally similar to the stacked X-structure (Duckett et al. 1995a), and the crystal structure of the DNAzyme shows that the stacked X-conformation can include one helical pair in the A-conformation (Nowakowski et al. 1999). However, modelling suggests that the juxtaposition of strands and grooves will be less satisfactory in RNA, and the higher magnesium ion concentration required to fold the RNA junction indicates a lower stability of the antiparallel form. Perhaps the biggest difference between the properties of the DNA and RNA four-way junctions is the lack of an unstacked structure at low salt concentrations for the RNA species. This clearly reflects a major difference in the electrostatic interactions in the RNA junction. In general the folding of branched DNA provides some good indications on the likely folding of the corresponding RNA species, but caution is required in making the extrapolation because the two polymers are significantly different. A number of studies point to the flexibility and malleability of branched nucleic acids, and this turns out to have particular significance in their interactions with proteins. Proteins such as the DNA junction-resolving enzymes exhibit considerable selectivity for the structure of their substrates, which is still not understood at a molecular level. Despite this, it appears to be universally true that these proteins distort the global, and in some cases at least the local, structure of the junctions. The somewhat perplexing result is that the proteins appear to distort the very property that they recognise. In general it seems that four-way DNA junctions are opened to one extent or another by interaction with proteins. (ABSTRACT TRUNCATED)  相似文献   

13.
Mutational, kinetic, and chemical modification experiments show that one of the three-way helical junctions in the Neurospora VS ribozyme contains a uridine turn that is important for organizing the functional three-dimensional structure of this junction. Disruption of the uridine turn disrupts the structure of the junction and decreases the self-cleavage activity of the ribozyme; however, substitution of the uridine turn with a variety of different hairpins, thereby transforming the three-way junction into a four-way junction, maintains catalytic activity. Chemical modification structure probing reveals that both the native junction and the hairpin-containing junction support the same tertiary interactions required elsewhere in the ribozyme for catalysis. These observations show that functionally equivalent three-dimensional RNA structures can be built from different secondary structure elements.  相似文献   

14.
RNA secondary structures can be divided into helical regions composed of canonical Watson-Crick and related base pairs, as well as single-stranded regions such as hairpin loops, internal loops, and junctions. These elements function as building blocks in the design of diverse RNA molecules with various fundamental functions in the cell. To better understand the intricate architecture of three-dimensional (3D) RNAs, we analyze existing RNA four-way junctions in terms of base-pair interactions and 3D configurations. Specifically, we identify nine broad junction families according to coaxial stacking patterns and helical configurations. We find that helices within junctions tend to arrange in roughly parallel and perpendicular patterns and stabilize their conformations using common tertiary motifs such as coaxial stacking, loop-helix interaction, and helix packing interaction. Our analysis also reveals a number of highly conserved base-pair interaction patterns and novel tertiary motifs such as A-minor-coaxial stacking combinations and sarcin/ricin motif variants. Such analyses of RNA building blocks can ultimately help in the difficult task of RNA 3D structure prediction.  相似文献   

15.
Structures of bulged three-way DNA junctions.   总被引:5,自引:3,他引:2       下载免费PDF全文
We have studied a series of three-way DNA junctions containing unpaired bases on one strand at the branch-point of the junctions. The global conformation of the arms of the junctions has been analysed by means of polyacrylamide gel electrophoresis, as a function of conditions. We find that in the absence of added metal ions, all the results for all the junctions can be accounted for by extended structures, with the largest angle being that between the arms defined by the strand containing the extra bases. Upon addition of magnesium (II) or hexamine cobalt (III) ions, the electrophoretic patterns change markedly, indicative of ion-dependent folding transitions for some of the junctions. For the junction lacking the unpaired bases, the three inter-arm angles appear to be quite similar, suggesting an extended structure. However, the addition of unpaired bases permits the three-way junction to adopt a significantly different structure, in which one angle becomes smaller than the other two. These species also exhibit marked protection against osmium addition to thymine bases at the point of strand exchange. These results are consistent with a model in which two of the helical arms undergo coaxial stacking in the presence of magnesium ions, with the third arm defining an angle that depends upon the number of unpaired bases.  相似文献   

16.
We present new modifications to the Wuchty algorithm in order to better define and explore possible conformations for an RNA sequence. The new features, including parallelization, energy-independent lonely pair constraints, context-dependent chemical probing constraints, helix filters, and optional multibranch loops, provide useful tools for exploring the landscape of RNA folding. Chemical probing alone may not necessarily define a single unique structure. The helix filters and optional multibranch loops are global constraints on RNA structure that are an especially useful tool for generating models of encapsidated viral RNA for which cryoelectron microscopy or crystallography data may be available. The computations generate a combinatorially complete set of structures near a free energy minimum and thus provide data on the density and diversity of structures near the bottom of a folding funnel for an RNA sequence. The conformational landscapes for some RNA sequences may resemble a low, wide basin rather than a steep funnel that converges to a single structure.  相似文献   

17.
The proton NMR spectra of DNA three-way junction complexes (TWJ) having unpaired pyrimidines, 5'-TT- and 5'-TC- on one strand at the junction site were assigned from 2D NOESY spectra acquired in H2O and D2O solvents and homonuclear 3D NOESY-TOCSY and 3D NOESY-NOESY in D2O solvent. TWJ are the simplest branched structures found in biologically active nucleic acids. Unpaired nucleotides are common features of such structures and have been shown to stabilize junction formation. The NMR data confirm that the component oligonucleotides assemble to form conformationally homogeneous TWJ complexes having three double-helical, B-form arms. Two of the helical arms stack upon each other. The unpaired pyrimidine bases lie in the minor groove of one of the helices and are partly exposed to solvent. The coaxial stacking arrangement deduced is different from that determined by Rosen and Patel (Rosen, M.A., and D.J. Patel. 1993. Biochemistry. 32:6576-6587) for a DNA three-way junction having two unpaired cytosines, but identical to that suggested by Welch et al. (Welch, J. B., D. R. Duckett, D. M. J. Lilley. 1993. Nucleic Acids Res. 21:4548-4555) on the basis of gel electrophoretic studies of DNA three-way junctions containing unpaired adenosines and thymidines.  相似文献   

18.
The packaging RNA (pRNA) found in phi29 bacteriophage is an essential component of a molecular motor that packages the phage''s DNA genome. The pRNA forms higher-order multimers by intermolecular “kissing” interactions between identical molecules. The phi29 pRNA is a proven building block for nanotechnology and a model to explore the rare phenomenon of naturally occurring RNA self-association. Although the self-association properties of the phi29 pRNA have been extensively studied and this pRNA is used in nanotechnology, the characteristics of phylogenetically related pRNAs with divergent sequences are comparatively underexplored. These diverse pRNAs may lend new insight into both the rules governing RNA self-association and for RNA engineering. Therefore, we used a combination of biochemical and biophysical methods to resolve ambiguities in the proposed secondary structures of pRNAs from M2, GA1, SF5, and B103 phage, and to discover that different naturally occurring pRNAs form multimers of different stoichiometry and thermostability. Indeed, the M2 pRNA formed multimers that were particularly thermostable and may be more useful than phi29 pRNA for many applications. To determine if diverse pRNA behaviors are conferred by different kissing loop sequences, we designed and tested chimeric RNAs based on our revised secondary structural models. We found that although the kissing loops are essential for self-association, the critical determinant of multimer stability and stoichiometry is likely the diverse three-way junctions found in these RNAs. Using known features of RNA three-way junctions and solved structures of phi29 pRNA''s junction, we propose a model for how different junctions affect self-association.  相似文献   

19.
Electrophoresis in polyacrylamide gels provides a simple yet powerful means of analyzing the relative disposition of helical arms in branched nucleic acids. The electrophoretic mobility of DNA or RNA with a central discontinuity is determined by the angle subtended between the arms radiating from the branchpoint. In a multi-helical branchpoint, comparative gel electrophoresis can provide a relative measure of all the inter-helical angles and thus the shape and symmetry of the molecule. Using the long-short arm approach, the electrophoretic mobility of all the species with two helical arms that are longer than all others is compared. This can be done as a function of conditions, allowing the analysis of ion-dependent folding of branched DNA and RNA species. Notable successes for the technique include the four-way (Holliday) junction in DNA and helical junctions in functionally significant RNA species such as ribozymes. Many of these structures have subsequently been proved correct by crystallography or other methods, up to 10 years later in the case of the Holliday junction. Just as important, the technique has not failed to date. Comparative gel electrophoresis can provide a window on both fast and slow conformational equilibria such as conformer exchange in four-way DNA junctions. But perhaps the biggest test of the approach has been to deduce the structures of complexes of four-way DNA junctions with proteins. Two recent crystallographic structures show that the global structures were correctly deduced by electrophoresis, proving the worth of the method even in these rather complex systems. Comparative gel electrophoresis is a robust method for the analysis of branched nucleic acids and their complexes.  相似文献   

20.
The structure of a large nucleic acid complex formed by the 10-23 DNA enzyme bound to an RNA substrate was determined by X-ray diffraction at 3.0 A resolution. The 82-nucleotide complex contains two strands of DNA and two strands of RNA that form five double-helical domains. The spatial arrangement of these helices is maintained by two four-way junctions that exhibit extensive base-stacking interactions and sharp turns of the phosphodiester backbone stabilized by metal ions coordinated to nucleotides at these junctions. Although it is unlikely that the structure corresponds to the catalytically active conformation of the enzyme, it represents a novel nucleic acid fold with implications for the Holliday junction structure.  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