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1.
J L Jestin  E Dème    A Jacquier 《The EMBO journal》1997,16(10):2945-2954
Thus far, conventional biophysical techniques, such as NMR spectroscopy or X-ray crystallography, allow the determination, at atomic resolution, of only structural domains of large RNA molecules such as group I introns. Determination of their overall spatial organization thus still relies on modeling. This requires that a relatively high number of tertiary interactions are defined in order to get sufficient topological constraints. Here, we report the use of a modification interference assay to identify structural elements involved in interdomain interactions. We used this technique, in a group II intron, to identify the elements involved in the interactions between domain V and the rest of the molecule. Domain V contains many of the active site components of these ribozymes. In addition to a previously identified 11 nucleotide motif involved in the binding of the domain V terminal GAAA tetraloop, a small number of elements were shown to be essential for domain V binding. In particular, we show that domain III is specifically required for the interaction with sequences encompassing the conserved 2 nucleotide bulge of domain V.  相似文献   

2.
The evolution of the group II intron in the plastid gene encoding tRNA(Val)UAC (trnV) from seven plant taxa was studied by aligning secondary and other structural features. Levels of evolutionary divergence between six angiosperms and a liverwort, Marchantia polymorpha, were compared for the six domains commonly demonstrated for group II introns and were shown to be statistically heterogeneous. Evolutionary rates varied substantially among various domains and other features. Domain II showed the highest evolutionary rate, approaching the synonymous substitution rate reported for cpDNA-encoded genes, while domain VI and the helix and loop region bearing EBS1 evolved at rates similar to those for nonsynonymous substitutions of a number of cpDNA-encoded genes. The minimum free-energy structure of domain I varied among the seven taxa, suggesting that possible protein-RNA or tertiary interactions are important for intron processing.  相似文献   

3.
The model describing the structure and conformational preferences of the HIV-Haiti V3 loop in the geometric spaces of Cartesian coordinates and dihedral angles was generated in terms of NMR spectroscopy data published in literature. To this end, the following successive steps were put into effect: (i) the NMR-based 3D structure for the HIV-Haiti V3 loop in water was built by computer modeling methods; (ii) the conformations of its irregular segments were analyzed and the secondary structure elements identified; and (iii) to reveal a common structural motifs in the HIV-Haiti V3 loop regardless of its environment variability, the simulated structure was collated with the one deciphered previously for the HIV-Haiti V3 loop in a water/trifluoroethanol (TFE) mixed solvent. As a result, the HIV-Haiti V3 loop was found to offer the highly variable fragment of gp120 sensitive to its environment whose changes trigger the large-scale structural rearrangements, bringing in substantial altering the secondary and tertiary structures of this functionally important site of the virus envelope. In spite of this fact, over half of amino acid residues that reside, for the most part, in the functionally important regions of the gp120 protein and may present promising targets for AIDS drug researches, were shown to preserve their conformational states in the structures under review. In particular, the register of these amino acids holds Asn-25 that is critical for the virus binding with primary cell receptor CD4 as well as Arg-3 that is critical for utilization of CCR5 co-receptor and heparan sulfate proteoglycans. The conservative structural motif embracing one of the potential sites of the gp120 N-linked glycosylation was detected, which seems to be a promising target for the HIV-1 drug design. The implications are discussed in conjunction with the literature data on the biological activity of the individual amino acids for the HIV-1 gp120 V3 loop.  相似文献   

4.
Abstract

The model describing the structure and conformational preferences of the HIV-Haiti V3 loop in the geometric spaces of Cartesian coordinates and dihedral angles was generated in terms of NMR spectroscopy data published in literature. To this end, the following successive steps were put into effect: (i) the NMR-based 3D structure for the HIV-Haiti V3 loop in water was built by computer modeling methods; (ii) the conformations of its irregular segments were analyzed and the secondary structure elements identified; and (iii) to reveal a common structural motifs in the HIV-Haiti V3 loop regardless of its environment variability, the simulated structure was collated with the one deciphered previously for the HIV-Haiti V3 loop in a water/trifluoroethanol (TFE) mixed solvent.

As a result, the HIV-Haiti V3 loop was found to offer the highly variable fragment of gp120 sensitive to its environment whose changes trigger the large-scale structural rearrangements, bringing in substantial altering the secondary and tertiary structures of this functionally important site of the virus envelope. In spite of this fact, over half of amino acid residues that reside, for the most part, in the functionally important regions of the gp120 protein and may present promising targets for AIDS drug researches, were shown to preserve their conformational states in the structures under review. In particular, the register of these amino acids holds Asn-25 that is critical for the virus binding with primary cell receptor CD4 as well as Arg-3 that is critical for utilization of CCR5 co-receptor and heparan sulfate proteoglycans. The conservative structural motif embracing one of the potential sites of the gp120 N-linked glycosylation was detected, which seems to be a promising target for the HIV-1 drug design.

The implications are discussed in conjunction with the literature data on the biological activity of the individual amino acids for the HIV-1 gp120 V3 loop.  相似文献   

5.
Group II introns are some of the largest ribozymes in nature, and they are a major source of information about RNA assembly and tertiary structural organization. These introns are of biological significance because they are self-splicing mobile elements that have migrated into diverse genomes and played a major role in the genomic organization and metabolism of most life forms. The tertiary structure of group II introns has been the subject of many phylogenetic, genetic, biochemical and biophysical investigations, all of which are consistent with the recent crystal structure of an intact group IIC intron from the alkaliphilic eubacterium Oceanobacillus iheyensis. The crystal structure reveals that catalytic intron domain V is enfolded within the other intronic domains through an elaborate network of diverse tertiary interactions. Within the folded core, DV adopts an activated conformation that readily binds catalytic metal ions and positions them in a manner appropriate for reaction with nucleic acid targets. The tertiary structure of the group II intron reveals new information on motifs for RNA architectural organization, mechanisms of group II intron catalysis, and the evolutionary relationships among RNA processing systems. Guided by the structure and the wealth of previous genetic and biochemical work, it is now possible to deduce the probable location of DVI and the site of additional domains that contribute to the function of the highly derived group IIB and IIA introns.  相似文献   

6.
Fedorova O  Pyle AM 《The EMBO journal》2005,24(22):3906-3916
Despite its importance for group II intron catalytic activity, structural information on conserved domain 3 (D3) is extremely limited. This domain is known to specifically stimulate the chemical rate of catalysis and to function as a 'catalytic effector'. Of all the long-range tertiary contacts that have been identified within group II introns, none has included D3 residues. Furthermore, little is known about the atoms and functional groups in D3 that contribute to catalysis. Using a nucleotide analog interference mapping assay with an extended repertoire of nucleotide analogs, we have identified functional groups in D3 that are critical for ribozyme activity. These data, together with mutational analysis, suggest the formation of noncanonical base pairs within the phylogenetically conserved internal loop at the base of D3. Finally, a related nucleotide analog interference suppression study resulted in the identification of a direct tertiary interaction between D3 and catalytic domain 5, which sheds new light on D3 function in the group II intron structure and mechanism.  相似文献   

7.
The intron sequence of chloroplast rpS16 and the secondary structure of its pre-mRNA were characterized for the first time in 26 Allium sativum accessions of different ecologo-geographical origins and seven related Allium species. The boundaries and main stem-loop consensus sequences were identified for all six domains of the intron. Polymorphism was estimated for the total intron and its regions. The structural regions of the rpS16 intron proved to be heterogeneous for mutation rate and spectrum. Mutations were most abundant in domains II and IV, and transition predominated in domains I, III, V, and VI. In addition to structural elements and motifs typical for group IIB introns, several Allium-specific micro- and macrostructural mutations were revealed. A 290-bp deletion involving domains III and IV and part of domain V was observed in A. altaicum, A. fistulosum, and A. schoenoprasum. Several indels and nucleotide substitutions were found to cause a deviation of the pre-mRNA secondary structure from the consensus model of group II introns.  相似文献   

8.
Group II introns are self-splicing, mobile genetic elements that have fundamentally influenced the organization of terrestrial genomes. These large ribozymes remain important for gene expression in almost all forms of bacteria and eukaryotes and they are believed to share a common ancestry with the eukaryotic spliceosome that is required for processing all nuclear pre-mRNAs. The three-dimensional structure of a group IIC intron was recently determined by X-ray crystallography, making it possible to visualize the active site and the elaborate network of tertiary interactions that stabilize the molecule. Here we describe the molecular features of the active site in detail and evaluate their correspondence with prior biochemical, genetic, and phylogenetic analyses on group II introns. In addition, we evaluate the structural significance of RNA motifs within the intron core, such as the major-groove triple helix and the domain 5 bulge. Having combined what is known about the group II intron core, we then compare it with known structural features of U6 snRNA in the eukaryotic spliceosome. This analysis leads to a set of predictions for the molecular structure of the spliceosomal active site.  相似文献   

9.
Despite the central role of group II introns in eukaryotic gene expression and their importance as biophysical and evolutionary model systems, group II intron tertiary structure is not well understood. In order to characterize the architectural organization of intron ai5gamma, we incorporated the photoreactive nucleotides s(4)U and s(6)dG at specific locations within the intron core and monitored the formation of cross-links in folded complexes. The resulting data reveal the locations for many of the most conserved, catalytically important regions of the intron (i.e., the J2/3 linker region, the IC1(i-ii) bulge in domain 1, the bulge of D5, and the 5'-splice site), showing that all of these elements are closely colocalized. In addition, we show by nucleotide analog interference mapping (NAIM) that a specific functional group in J2/3 plays a role in first-step catalysis, which is consistent with its apparent proximity to other first-step components. These results extend our understanding of active-site architecture during the first step of group II intron self-splicing and they provide a structural basis for spliceosomal comparison.  相似文献   

10.
Comparative and functional anatomy of group II catalytic introns--a review   总被引:88,自引:0,他引:88  
F Michel  K Umesono  H Ozeki 《Gene》1989,82(1):5-30
The 70 published sequences of group II introns from fungal and plant mitochondria and plant chloroplasts are analyzed for conservation of primary sequence, secondary structure and three-dimensional base pairings. Emphasis is put on structural elements with known or suspected functional significance with respect to self-splicing: the exon-binding and intron-binding sites, the bulging A residue involved in lariat formation, structural domain V and two isolated base pairs, one of them involving the last intron nucleotide and the other one, the first nt of the 3' exon. Separate sections are devoted to the 29 group II-like introns from Euglena chloroplasts and to the possible relationship of catalytic group II introns to nuclear premessenger introns. Alignments of all available sequences of group II introns are provided in the APPENDIX.  相似文献   

11.
Group II introns are self-splicing RNA molecules that also behave as mobile genetic elements. The secondary structure of group II intron RNAs is typically described as a series of six domains that project from a central wheel. Most structural and mechanistic analyses of the intron have focused on domains 1 and 5, which contain the residues essential for catalysis, and on domain 6, which contains the branch-point adenosine. Domains 2 and 3 (D2, D3) have been shown to make important contributions to intronic activity; however, information about their function is quite limited. To elucidate the role of D2 and D3 in group II ribozyme catalysis, we built a series of multi-piece ribozyme constructs based on the ai5gamma group II intron. These constructs are designed to shed light on the roles of D2 and D3 in some of the major reactions catalyzed by the intron: 5'-exon cleavage, branching, and substrate hydrolysis. Reactions with these constructs demonstrate that D3 stimulates the chemical rate constant of group II intron reactions, and that it behaves as a form of catalytic effector. However, D3 is unable to associate independently with the ribozyme core. Docking of D3 is mediated by a short duplex that is found at the base of D2. In addition to recruiting D3 into the core, the D2 stem directs the folding of the adjacent j(2/3) linker, which is among the most conserved elements in the group II intron active site. In turn, the D2 stem contributes to 5'-splice site docking and ribozyme conformational change. Nucleotide analog interference mapping suggests an interaction between the D2 stem and D3 that builds on the known theta-theta' interaction and extends it into D3. These results establish that D3 and the base of D2 are key elements of the group II intron core and they suggest a hierarchy for active-site assembly.  相似文献   

12.
Group II intron domain 5 facilitates a trans-splicing reaction.   总被引:34,自引:3,他引:31       下载免费PDF全文
A self-splicing group II intron of yeast mitochondrial DNA (aI5g) was divided within intron domain 4 to yield two RNAs that trans-spliced in vitro with associated trans-branching of excised intron fragments. Reformation of the domain 4 secondary structure was not necessary for the trans reaction, since domain 4 sequences were shown to be dispensable. Instead, the trans reaction depended on a previously unpredicted interaction between intron domain 5, the most highly conserved region of group II introns, and another region of the RNA. Domain 5 was shown to be essential for cleavage at the 5' splice site. It stimulated that cleavage when supplied as a trans-acting RNA containing only 42 nucleotides of intron sequence. The relevance of our findings to in vivo trans-splicing mechanisms is discussed.  相似文献   

13.
The conformation of Escherichia coli 5 S rRNA was investigated using chemical and enzymatic probes. The four bases were monitored at one of their Watson-Crick positions with dimethylsulfate (at C(N-3) and A(N-1], with a carbodiimide derivative (at G(N-1) and U(N-3] and with kethoxal (at G(N-1, N-2]. Position N-7 of purine was probed with diethylpyrocarbonate (at A(N-7] and dimethylsulfate (at G(N-7]. Double-stranded or stacked regions were tested with RNase V1 and unpaired guanine residues with RNase T1. We also used lead(II) that has a preferential affinity for interhelical and loop regions and a high sensitivity for flexible regions. Particular care was taken to use uniform conditions of salt, magnesium, pH and temperature for the different enzymatic chemical probes. Derived from these experimental data, a three dimensional model of the 5 S rRNA was built using computer modeling which integrates stereochemical constraints and phylogenetic data. The three domains of 5 S rRNA secondary structure fold into a Y-shaped structure that does not accommodate long-range tertiary interactions between domains. The three domains have distinct structural and dynamic features as revealed by the chemical reactivity and the lead(II)-induced hydrolysis: domain 2 (loop B/helix III/loop C) displays a rather weak structure and possesses dynamic properties while domain 3 (helix V/region E/helix IV/loop D) adopts a highly structured and overall helical conformation. Conserved nucleotides are not crucial for the tertiary folding but maintain an intrinsic structure in the loop regions, especially via non-canonical pairing (A.G, G.U, G.G, A.C, C.C), which can close the loops in a highly specific fashion. In particular, nucleotides in the large external loop C fold into an organized conformation leading to the formation of a five-membered loop motif. Finally, nucleotides at the hinge region of the Y-shape are involved in a precise array of hydrogen bonds based on a triple interaction between U14, G69 and G107 stabilizing the quasi-colinearity of helices II and V. The proposed tertiary model is consistent with the localization of the ribosomal protein binding sites and possesses strong analogy with the model proposed for Xenopus laevis 5 S rRNA, indicating that the Y-shape model can be generalized to all 5 S rRNAs.  相似文献   

14.
Coxsackievirus B3 (CVB3) is a picornavirus which causes myocarditis and pancreatitis and may play a role in type I diabetes. The viral genome is a single 7,400-nucleotide polyadenylated RNA encoding 11 proteins in a single open reading frame. The 5' end of the viral genome contains a highly structured nontranslated region (5'NTR) which folds to form an internal ribosome entry site (IRES) as well as structures responsible for genome replication, both of which are critical for virulence. A structural model of the CVB3 5'NTR, generated primarily by comparative sequence analysis and energy minimization, shows seven domains (I to VII). While this model provides a preliminary basis for structural analysis, the model lacks comprehensive experimental validation. Here we provide experimental evidence from chemical modification analysis to determine the structure of the CVB3 5'NTR. Chemical probing results show that the theoretical model for the CVB3 5'NTR is largely, but not completely, supported experimentally. In combination with our chemical probing data, we have used the RNASTRUCTURE algorithm and sequence comparison of 105 enterovirus sequences to provide evidence for novel secondary and tertiary interactions. A comprehensive examination of secondary structure is discussed, along with new evidence for tertiary interactions. These include a loop E motif in domain III and a long-range pairing interaction that links domain II to domain V. The results of our work provide mechanistic insight into key functional elements in the cloverleaf and IRES, thereby establishing a base of structural information from which to interpret experiments with CVB3 and other picornaviruses.  相似文献   

15.
16.
Frequent use of the same tertiary motif by self-folding RNAs.   总被引:28,自引:7,他引:21       下载免费PDF全文
M Costa  F Michel 《The EMBO journal》1995,14(6):1276-1285
We have identified an 11 nucleotide RNA motif, [CCUAAG...UAUGG], that is extraordinarily abundant in group I and group II self-splicing introns at sites known, or suspected from co-variation analysis, to interact with hairpin terminal loops with a GNRA consensus sequence. Base substitution experiments using a ribozyme-substrate system derived from a group I intron reveal that this motif interacts preferentially with GAAA terminal loops and binds them with remarkable affinity, compared with other known combinations of GNRA loops and matched targets. A copy of the [CCUAAG...UAUGG] motif which is present in domain I of many group II introns is shown to interact with the GAAA terminal loop that caps domain V. This is the first interaction to be identified between these two domains, whose mutual recognition is known to be necessary and sufficient for group II ribozymic activity. We conclude that interaction of [CCUAAG...UAUGG] with GAAA loops is one of the most common solutions used by nature to solve the problem of compacting and bringing together RNA structural domains.  相似文献   

17.
The X-ray crystal structure of an excised group II self-splicing intron was recently solved by the Pyle group. Here we review some of the notable features of this structure and what they may tell us about the catalytic active site of the group II ribozyme and potentially the spliceosome. The new structure validates the central role of domain V in both the structure and catalytic function of the ribozyme and resolves several outstanding puzzles raised by previous biochemical, genetic and structural studies. While lacking both exons as well as the cleavage sites and nucleophiles, the structure reveals how a network of tertiary interactions can position two divalent metal ions in a configuration that is ideal for catalysis.  相似文献   

18.
The model describing the conformational properties of the HIV-1 principal neutralizing determinant in the geometric space of dihedrals was generated in terms of NMR spectroscopy data published in literature. To gain an object in view, the following successive steps were put into effect: (i) the NMR-based local structures for the HIV(MN) V3 loop were determined in water and in a mixed water/trifluoroethanol (TFE) solvent (7:3), (ii) in either case, the conformations of its irregular segments were analyzed and the secondary structure elements identified, (iii) to appreciate the degree of conformational mobility of the stretch of interest, the simulated structures were compared with each other, (iv) to detect the amino acids retaining their conformations inside the diverse HIV-1 isolates, the structures computed were collated with the one derived previously for the V3 loop from Thailand isolate, and (v) as a matter of record, the structurally rigid residues, that may present the forward-looking targets for AIDS drug researches, were revealed. Summing up the principal results arising from these studies, the following conclusions were drawn: I. The HIV(MN) V3 loop offers the highly mobile fragment of gp120 sensitive to its environment whose changes trigger the large-scale structural reforms, bringing in substantial altering the secondary structure of this functionally important site of the virus envelope. II. In water, it exhibits extended site 1-14 separated by double beta-turn 15-20 with unordered region 21-35. III. Adding the TFE gives rise to destruction of the regular structure in the V3 loop N-terminal, stimulates the formation of 3(10)-helix in site 24-31, and affects also its central region 20-25 forming the HIV-1 immunogenic crown. IV. Regardless of statistically significant differences between local structures of the HIV(MN) V3 loop in water and in water/TFE solution, over one-third of residues keeps their conformational states; the register of these amino acids comprises Asn-25 critical for virus binding with primary cell receptor CD4 as well as Arg-3 critical for utilization of CCR5 coreceptor. V. There are no conserved structural motifs within the V3 loops from Minnesota and Thailand HIV-1 strains. However, perceptible portion of amino acids (more than 35%), including those appearing in the functionally important regions of gp120, holds the values of dihedral angles in which case. The implications are discussed in conjunction with the data on the experimental observations for the HIV-1 principal neutralizing determinant.  相似文献   

19.
The sequences of several members of the myosin family of molecular motors are evaluated using ASP (Ambivalent Structure Predictor), a new computational method. ASP predicts structurally ambivalent sequence elements by analyzing the output from a secondary structure prediction algorithm. These ambivalent sequence elements form secondary structures that are hypothesized to function as switches by undergoing conformational rearrangement. For chicken skeletal muscle myosin, 13 discrete structurally ambivalent sequence elements are identified. All 13 are located in the heavy chain motor domain. When these sequence elements are mapped into the myosin tertiary structure, they form two compact regions that connect the actin binding site to the adenosine 5'-triphosphate (ATP) site, and the ATP site to the fulcrum site for the force-producing bending of the motor domain. These regions, predicted by the new algorithm to undergo conformational rearrangements, include the published known and putative switches of the myosin motor domain, and they form plausible allosteric connections between the three main functional sites of myosin. The sequences of several other members of the myosin I and II families are also analyzed.  相似文献   

20.

Background

Group II introns are mobile genetic elements that form conserved secondary and tertiary structures. In order to determine which of the conserved structural elements are required for mobility, a series of domain and sub-domain deletions were made in the Lactococcus lactis group II intron (Ll.LtrB) and tested for mobility in a genetic assay. Point mutations in domains V and VI were also tested.

Results

The largest deletion that could be made without severely compromising mobility was 158 nucleotides in DIVb(1–2). This mutant had a mobility frequency comparable to the wild-type Ll.LtrB intron (ΔORF construct). Hence, all subsequent mutations were done in this mutant background. Deletion of DIIb reduced mobility to approximately 18% of wild-type, while another deletion in domain II (nts 404–459) was mobile to a minor extent. Only two deletions in DI and none in DIII were tolerated. Some mobility was also observed for a DIVa deletion mutant. Of the three point mutants at position G3 in DV, only G3A retained mobility. In DVI, deletion of the branch-point nucleotide abolished mobility, but the presence of any nucleotide at the branch-point position restored mobility to some extent.

Conclusions

The smallest intron capable of efficient retrohoming was 725 nucleotides, comprising the DIVb(1–2) and DII(ii)a,b deletions. The tertiary elements found to be nonessential for mobility were alpha, kappa and eta. In DV, only the G3A mutant was mobile. A branch-point residue is required for intron mobility.  相似文献   

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