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1.
Conserved sequences and structures of group I introns: building an active site for RNA catalysis--a review 总被引:51,自引:0,他引:51
Group I introns fold to form an active site to mediate their own RNA splicing. Sequence elements conserved among the available set of 66 group I introns are compiled. Comparative sequence analysis leads to the prediction of some conserved structural features that have not been widely appreciated. The possible significance of conserved nucleotides within base-paired duplexes is discussed; they might be involved in base triplets or alternate pairing interactions. 相似文献
2.
Forty-eight RNA duplexes were constructed that contained all common single base bulges at six different locations. The stabilities of the RNAs were determined by temperature gradient gel electrophoresis (TGGE). The relative stability of a single base bulge was dependent on both base identity and the nearest neighbor context. The single base bulges were placed into two categories. A bulged base with no identical neighboring base was defined as a Group I base bulge. Group II-bulged bases had at least one neighboring base identical to it. Group II bulges were generally more stable than Group I bulges in the same nearest neighbor environments. This indicates that position degeneracy of an unpaired base enhances stability. Differences in the mobility transition temperatures between the RNA fragments with bulges and the completely base-paired reference RNAs were related to free energy differences. Simple models for estimating the free energy contribution of single base bulges were evaluated from the free energy difference data. The contribution of a Group I bulge 5'-(XNZ)-3'.5'-(Z'-X')-3' where N is the unpaired base and X.X' and Z.Z' the neighboring base pairs, could be well-represented (+/-0.34 kcal/mol) by the equation, DeltaG((X)(N)()(Z))(.)((Z)(')(-)(X)(')()) = 3.11 + 0. 40DeltaG(s)()((XZ))(.)((Z)(')(X)(')()). DeltaG(s)()((XZ))(. )((Z)(')(X)(')()) is the stacking energy of the closing base pair doublet. By adding a constant term, delta = -0.3 kcal/mol, to the right side of the above equation, free energies of Group II bulges could also be predicted with the same accuracy. The term delta represents the stabilizing effect due to position degeneracy. A similar equation/model was applied to previous data from 32 DNA fragments with single base bulges. It predicted the free energy differences with a similar standard deviation. 相似文献
3.
Synthesis and properties of oligonucleotides containing 2''-deoxynebularine and 2''-deoxyxanthosine. 总被引:9,自引:5,他引:4
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R Eritja D M Horowitz P A Walker J P Ziehler-Martin M S Boosalis M F Goodman K Itakura B E Kaplan 《Nucleic acids research》1986,14(20):8135-8153
The preparation of synthetic oligonucleotides containing 2'-deoxynebularine (dN) and 2'-deoxyxanthosine (dX) is described. The thermal stabilities of duplexes containing dX, dN, and 2'-deoxyinosine (dI) base-paired with the four natural bases have been measured. Xanthine base pairs have stabilities at pH 5.5 that are similar to those of dI-containing duplexes at neutral pH. When xanthine is paired with adenine or cytosine an unusual stabilization of the duplex structure is observed at acid pH. Incorporation of base mispairs opposite template xanthine sites were measured using Drosophila DNA polymerase alpha. The relative nucleoside incorporation rates are in the order: T greater than C much greater than A approximately equal to G. These rates do not correlate with relative thermodynamic stabilities of base mispairs with xanthine obtained from Tm measurements: T greater than G greater than A approximately equal to C. We suggest that DNA polymerase misinsertion rates are greatest when the base mispair can be formed in accordance with Watson-Crick as opposed to other base pairing geometries even though other geometries, e.g. wobble, may result in a more stable final DNA product. 相似文献
4.
The three-dimensional solution structure of a DNA molecule of the sequence 5'-d(GCATCGAAAAAGCTACG)-3' paired with 5'-d(CGTAGCCGATGC)-3' containing a five-adenine bulge loop (dA(5)-bulge) between two double helical stems was determined by 2D (1)H and (31)P NMR, infrared, and Raman spectroscopy. The DNA in both stems adopt a classical B-form double helical structure with Watson-Crick base pairing and C2'-endo sugar conformation. In addition, the two dG/dC base pairs framing the dA(5)-bulge loop are formed and are stable at least up to 30 degrees C. The five adenine bases of the bulge loop are localized at intrahelical positions within the double helical stems. Stacking on the double helical stem is continued for the first four 5'-adenines in the bulge loop. The total rise (the height) of these four stacked adenines roughly equals the diameter of the double helical stem. The stacking interactions are broken between the last of these four 5'-adenines and the fifth loop adenine at the 3'-end. This 3'-adenine partially stacks on the other stem. The angle between the base planes of the two nonstacking adenines (A10 and A11) in the bulge loop reflects the kinking angle of the global DNA structure. The neighboring cytosines opposite the dA(5)-bulge (being parts of the bulge flanking base pairs) do not stack on one another. This disruption of stacking is characterized by a partial shearing of these bases, such that certain sequential NOEs for this base step are preserved. In the base step opposite the loop, an extraordinary hydrogen bond is observed between the phosphate backbone of the 5'-dC and the amino proton of the 3'-dC in about two-thirds of the conformers. This hydrogen bond probably contributes to stabilizing the global DNA structure. The dA(5)-bulge induces a local kink into the DNA molecule of about 73 degrees (+/-11 degrees ). This kinking angle and the mutual orientation of the two double helical stems agree well with results from fluorescence resonance energy transfer measurements of single- and double-bulge DNA molecules. 相似文献
5.
We have synthesized an oligodeoxynucleotide duplex, d(G-C-A-T-C-G-A-T-A-G-C-T-A-C-G).d(C-G-T-A-G-C-C-G-A-T-C-G), with a three-base bulge loop (A-T-A) at a central site in the first strand. Nuclear Overhauser experiments (NOESY) in H2O indicate that the GC base pairs flanking the bulge loop are intact between 0 and 25 degrees C. Nuclear Overhauser effects in both H2O and D2O indicate that all bases within the bulge loop are stacked into the helix. These unpaired bases retain an anti conformation about their glycosidic bonds as they stack within the duplex. The absence of normal sequential connectivities between the two cytosine residues flanking the bulge site on the opposite strand indicates a disruption in the geometry of this base step upon insertion of the bulged bases into the helix. This conformational perturbation is more akin to a shearing apart of the bases, which laterally separates the two halves of the molecule, rather than the "wedge" model often invoked for single-base bulges. Using molecular dynamics calculations, with both NOE-derived proton-proton distances and relaxation matrix-calculated NOESY cross peak volumes as restraints, we have determined the solution structure of an A-T-A bulge loop within a DNA duplex. The bulged bases are stacked among themselves and with the guanine bases on either side of the loop. All three of the bulged bases are displaced by 2-3 A into the major groove, increasing the solvent accessibility of these residues. The ATA-bulge duplex is significantly kinked at the site of the lesion, in agreement with previously reported electron microscopy and gel retardation studies on bulge-containing duplexes [Hsieh, C.-H., & Griffith, J. D. (1989) Proc. Natl. Acad. Sci. U.S.A 86, 4833-4837; Bhattacharyya, A., & Lilley, D. M. J. (1989) Nucleic Acids Res. 17, 6821-6840]. Bending occurs in a direction away from the bulge-containing strand, and we find a significant twist difference of 84 degrees between the two base pairs flanking the bulge loop site. This value represents 58% of the twist difference for base pairs four steps apart in B-DNA. These results suggest a structural mechanism for the bending of DNA induced by unpaired bases, as well as accounting for the effect bulge loops may have on the secondary and tertiary structures of nucleic acids. 相似文献
6.
Assessment of a model for intron RNA secondary structure relevant to RNA self-splicing--a review 总被引:36,自引:0,他引:36
A widespread class of introns is characterized by a particular RNA secondary structure, based upon four conserved nucleotide sequences. Among such "class I" introns are found the majority of introns in fungal mitochondrial genes and the self-splicing intron of the large ribosomal RNA of several species of Tetrahymena. A model of the RNA secondary structure, which must underlie the self-splicing activity, is here evaluated in the light of data on 16 further introns. The main body or "core structure" of the intron always consists of the base-paired regions P3 to P9 with the associated single-stranded loops, with P2 present also in most cases. Two minority sub-classes of core structure occur, one of which is typical of introns in fungal ribosomal RNA. Introns in which the core structure is close to the 5' splice site all have an internal guide sequence (IGS) which can pair with exon sequences adjacent to the 5' and 3' splice sites to align them precisely, as proposed by Davies et al. [Nature 300 (1982) 719-724]. In these cases, the internal guide model allows us to predict correctly the exact location of splice sites. All other introns probably use other mechanisms of alignment. This analysis provides strong support for the RNA splicing model which we have developed. 相似文献
7.
S P Hwang M Eisenberg R Binder G S Shelness D L Williams 《The Journal of biological chemistry》1989,264(14):8410-8418
Analyses of apolipoprotein II mRNA with chemical and enzymatic probes showed that double- and single-stranded regions were distributed uniformly along the mRNA except for a large (72 nucleotides) single-stranded region containing the translation stop codon. Secondary structure models constrained by the experimental data were made by varying the distance (along the mRNA) over which base pairing was allowed. Four prominent secondary structures were seen with restrictions of 165, 330, or 659 nucleotides suggesting that such structures from via local interactions over distances of 50-120 nucleotides. Predicted long range interactions involve only 2-3 base pairs while local interactions involve helices of 4-10 base pairs. Predicted helices of greater than or equal to 4 base pairs occur primarily within exons, raising the possibility that prominent secondary structures in mRNAs may be largely due to intraexonic base pairing. Tests of single- and double-stranded domains by oligonucleotide-directed RNase H cleavage and primer extension were in accord with the structure model and with nuclease and chemical modification data. The model predicting base pairing between the coding and the 3' noncoding regions was tested by RNase H cleavage followed by oligo(dT)-cellulose chromatography to separate 5' and 3' mRNA fragments. Most (82%) of the 5' fragment remained associated with the 3' noncoding region in a structure with a tm = 50 degrees C in 0.2 M Na+ suggesting that this stem could be stable in vivo. This stem may be stable in the isolated mRNA, but would likely occur transiently in polyribosomal apolipoprotein II mRNA due to ribosome transit through the 5' side of the stem. Alternate structures may occur in this region during ribosome transit and play a role in translation termination or in determining the susceptibility of the mRNA to degradation. 相似文献
8.
9.
Effects of single-base bulges on intercalator binding to small RNA and DNA hairpins and a ribosomal RNA fragment 总被引:4,自引:0,他引:4
The way in which a single-base bulge might affect the structure of an RNA helix has been examined by preparing a series of six RNA hairpins, all with seven base pairs and a four-nucleotide loop. Five of the hairpins have single-base bulges at different positions. The intercalating cleavage reagent (methidiumpropyl)-EDTA-Fe(II) [MPE-Fe(II)] binds preferentially at a CpG sequence in the helix lacking a bulge and in four of the five hairpins with bulges. Hairpins with a bulge one or two bases to the 3' side of the CpG sequence bind ethidium 4-5-fold more strongly than the others. V1 RNase, which is sensitive to RNA backbone conformation in helices, detects a conformational change in all of the helices when ethidium binds; the most dramatic changes, involving the entire hairpin stem, are in one of the two hairpins with enhanced ethidium affinity. Only a slight conformational change is detected in the hairpin lacking a bulge. A bulge adjacent to a CpG sequence in a 100-nucleotide ribosomal RNA fragment enhances MPE-Fe(II) binding by an order of magnitude. These results extend our previous observations of bulges at a single position in an RNA hairpin [White, S. A., & Draper, D.E. (1987) Nucleic Acids Res. 15, 4049] and show that (1) a structural change in an RNA helix may be propagated for several base pairs, (2) bulges tend to increase the number of conformations available to a helix, and (3) the effects observed in small RNA hairpins are relevant to larger RNAs with more extensive structure. A bulge in a DNA hairpin identical in sequence with the RNA hairpins does not enhance MPE-Fe(II) binding affinity, relative to a control DNA hairpin. The effects of bulges on ethidium intercalation are evidently modulated by helix structure. 相似文献
10.
Effects of bulge composition and flanking sequence on the kinking of DNA by bulged bases 总被引:7,自引:0,他引:7
We recently showed that bulged bases kink duplex DNA, with the degree of kinking increasing in roughly equal increments as the number of bases in the bulge increases from one to four [Hsieh, C.-H., & Griffith, J.D. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 4833-4837]. Here we have examined the kinking of DNA by single A, C, G, or T bulges with different neighboring base pairs. Synthetic 30 base pair (bp) duplex DNAs containing 2 single-base bulges spaced by 10 bp were ligated head to tail, and their electrophoretic behavior in highly cross-linked gels was examined. All bulge-containing DNAs showed marked electrophoretic retardations as compared to non-bulge-containing DNA. Regardless of the sequence of the flanking base pairs, purine bulges produced greater retardations than pyrimidine bulges. Furthermore, C and T bulges produced the same retardations as did G and A bulges. Bulged DNA containing different flanking base pairs showed marked differences in electrophoretic mobility. For C-bulged DNA, the greatest retardations were observed with G.C neighbors, the least with T.A neighbors, and an intermediate amount with a mixture of neighboring base pairs. For A-bulged DNA, the retardations were greatest with G.C neighbors, less with T.A neighbors, even less with a mixture of neighboring base pairs, and finally least with C.G neighbors. Thus flanking base pairs affect C-bulged DNA and A-bulged DNA differently, and G.C and C.G flanking base pairs were seen to have very different effects. These results imply an important role of base stacking in determining how neighboring base pairs influence the kinking of DNA by a single-base bulge. 相似文献
11.
12.
Interphotoreceptor retinoid-binding protein. Gene characterization, protein repeat structure, and its evolution 总被引:8,自引:0,他引:8
D E Borst T M Redmond J E Elser M A Gonda B Wiggert G J Chader J M Nickerson 《The Journal of biological chemistry》1989,264(2):1115-1123
The gene for bovine interphotoreceptor retinoid-binding protein (IRBP) has been cloned, and its nucleotide sequence has been determined. The IRBP gene is about 11.6 kilobase pairs (kb) and contains four exons and three introns. It transcribed into a large mRNA of approximately 6.4 kb and translated into a large protein of 145,000 daltons. To prove the identity of the genomic clone, we determined the protein sequence of several tryptic and cyanogen bromide fragments of purified bovine IRBP protein and localized them in the protein predicted from its nucleotide sequence. There is a 4-fold repeat structure in the protein sequence with 30-40% sequence identity and many conservative substitutions between any two of the four protein repeats. The third and fourth repeats are the most similar pair. All three of the introns in the IRBP gene fall in the fourth protein repeat. Two of the exons, the first and the fourth, are large, 3173 and 2447 bases, respectively. The introns are each about 1.5-2.2 kb long. The human IRBP gene has a sequence that is similar to one of the introns from the bovine gene. The unexpected gene structure and protein repeat structure in the bovine gene lead us to propose a model for the evolution of the IRBP gene. 相似文献
13.
14.
Features of two hepatitis B virus (HBV) DNA integrations suggest mechanisms of HBV integration. 总被引:9,自引:2,他引:7
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Two integrated hepatitis B virus (HBV) DNA molecules were cloned from two primary hepatocellular carcinomas each containing only a single integration. One integration (C3) contained a single linear segment of HBV DNA, and the other integration (C4) contained a large inverted duplication of viral DNA at the site of a chromosome translocation (O. Hino, T.B. Shows, and C.E. Rogler, Proc. Natl. Acad. Sci. USA 83:8338-8342, 1986). Sequence analysis of the virus-cell junctions of C3 placed the left virus-cell junction at nucleotide 1824, which is at the 5' end of the directly repeated DR1 sequence and is 6 base pairs from the 3' end of the long (L) negative strand. The right virus-cell junction was at nucleotide 1762 in a region of viral DNA (within the cohesive overlap) which shared 5-base-pair homology with cellular DNA. Sequence analysis of the normal cellular DNA across the integration site showed that 11 base pairs of cellular DNA were deleted at the site of integration. On the basis of this analysis, we suggest a mechanism for integration of the viral DNA molecule which involves strand invasion of the 3' end of the L negative strand of an open circular or linear HBV DNA molecule (at the DR1 sequence) and base pairing of the opposite end of the molecule with cellular DNA, accompanied by the deletion of 11 base pairs of cellular DNA during the double recombination event. Sequencing across the inverted duplication of HBV DNA in clone C4 located one side of the inversion at nucleotide 1820, which is 2 base pairs from the 3' end of the L negative strand. Both this sequence and the left virus-cell junction of C3 are within the 9-nucleotide terminally redundant region of the HBV L negative strand DNA. We suggest that the terminal redundancy is a preferred topoisomerase I nicking region because of both its base sequence and forked structure. Such nicking would lead to integration and rearrangement of HBV molecules within the terminal redundancy, as we have observed in both our clones. 相似文献
15.
The ascomycetous fungus Cryptendoxyla hypophloia contains an insertion of 433 base pairs in the genes encoding nuclear small subunit ribosomal RNA. Secondary structure analyses
of the insert reveal characteristics indicative of a Group I intron, including elements P, Q, R, and S; however, the sequences
of these conserved regions deviate significantly from recognized consensus sequences for Group I introns. Principal-components
analysis, based on 79 nucleotide positions from the conserved core sequences of 93 Group I introns, identified 17 introns
similar to that of C. hypophloia. This grouping, which includes inserts from phylogenetically diverse organisms, cannot readily be classified in any previously
recognized major group of Group I introns. We propose the creation of a new group, IE, to accommodate these sequences, and
discuss the evolutionary relationships between group IE and other major groups of Group I introns.
Received: 11 January 1998 / Accepted: 12 October 1998 相似文献
16.
Recent studies have shown that topological constraints encoded at the RNA secondary structure level involving basic steric and stereochemical forces can significantly restrict the orientations sampled by helices across two-way RNA junctions. Here, we formulate these topological constraints in greater quantitative detail and use this topological framework to rationalize long-standing but poorly understood observations regarding the basic behavior of RNA two-way junctions. Notably, we show that the asymmetric nature of the A-form helix and the finite length of a bulge provide a physical basis for the experimentally observed directionality and bulge-length amplitude dependence of bulge induced inter-helical bends. We also find that the topologically allowed space can be modulated by variations in sequence, particularly with the addition of non-canonical GU base pairs at the junction, and, surprisingly, by the length of the 5' and 3' helices. A survey of two-way RNA junctions in the protein data bank confirms that junction residues have a strong preference to adopt looped-in, non-canonically base-paired conformations, providing a route for extending our bulge-directed framework to internal loop motifs and implying a simplified link between secondary and tertiary structure. Finally, our results uncover a new simple mechanism for coupling junction-induced topological constraints with tertiary interactions. 相似文献
17.
Solution structure of an RNA fragment with the P7/P9.0 region and the 3'-terminal guanosine of the tetrahymena group I intron
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Kitamura A Muto Y Watanabe S Kim I Ito T Nishiya Y Sakamoto K Ohtsuki T Kawai G Watanabe K Hosono K Takaku H Katoh E Yamazaki T Inoue T Yokoyama S 《RNA (New York, N.Y.)》2002,8(4):440-451
In the second step of the two consecutive transesterifications of the self-splicing reaction of the group I intron, the conserved guanosine at the 3' terminus of the intron (omegaG) binds to the guanosine-binding site (GBS) in the intron. In the present study, we designed a 22-nt model RNA (GBS/omegaG) including the GBS and omegaG from the Tetrahymena group I intron, and determined the solution structure by NMR methods. In this structure, omegaG is recognized by the formation of a base triple with the G264 x C311 base pair, and this recognition is stabilized by the stacking interaction between omegaG and C262. The bulged structure at A263 causes a large helical twist angle (40 +/- 80) between the G264 x C311 and C262 x G312 base pairs. We named this type of binding pocket with a bulge and a large twist, formed on the major groove, a "Bulge-and-Twist" (BT) pocket. With another twist angle between the C262 x G312 and G413 x C313 base pairs (45 +/- 100), the axis of GBS/omegaG is kinked at the GBS region. This kinked axis superimposes well on that of the corresponding region in the structure model built on a 5.0 A resolution electron density map (Golden et al., Science, 1998, 282:345-358). This compact structure of the GBS is also consistent with previous biochemical studies on group I introns. The BT pockets are also found in the arginine-binding site of the HIV-TAR RNA, and within the 16S rRNA and the 23S rRNA. 相似文献
18.
The gene coding for small ribosomal subunit RNA in the basidiomycete Ustilago maydis contains a group I intron.
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The nucleotide sequence of the gene coding for small ribosomal subunit RNA in the basidiomycete Ustilago maydis was determined. It revealed the presence of a group I intron with a length of 411 nucleotides. This is the third occurrence of such an intron discovered in a small subunit rRNA gene encoded by a eukaryotic nuclear genome. The other two occurrences are in Pneumocystis carinii, a fungus of uncertain taxonomic status, and Ankistrodesmus stipitatus, a green alga. The nucleotides of the conserved core structure of 101 group I intron sequences present in different genes and genome types were aligned and their evolutionary relatedness was examined. This revealed a cluster including all group I introns hitherto found in eukaryotic nuclear genes coding for small and large subunit rRNAs. A secondary structure model was designed for the area of the Ustilago maydis small ribosomal subunit RNA precursor where the intron is situated. It shows that the internal guide sequence pairing with the intron boundaries fits between two helices of the small subunit rRNA, and that minimal rearrangement of base pairs suffices to achieve the definitive secondary structure of the 18S rRNA upon splicing. 相似文献
19.
The hammerhead structural model proposed for sequences that mediate self-cleavage of certain RNAs contains base-paired three stems and 13 conserved bases. Insertion, deletion and base substitution mutations were carried out on a 58 base RNA containing the sequence of the single-hammerhead structure of the plus RNA of the virusoid of lucerne transient streak virus, and the effects on self-cleavage assessed. Results showed that there is flexibility in the sequence requirements for self-cleavage in vitro, but alterations of the conserved sequence or predicted secondary structure generally reduced the efficiency of self-cleavage. 相似文献
20.
Conserved nucleotides in the TAR RNA stem of human immunodeficiency virus type 1 are critical for Tat binding and trans activation: model for TAR RNA tertiary structure. 总被引:5,自引:0,他引:5
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U Delling L S Reid R W Barnett M Y Ma S Climie M Sumner-Smith N Sonenberg 《Journal of virology》1992,66(5):3018-3025
Interaction between the human immunodeficiency virus type 1 (HIV-1) trans-activator Tat and its cis-acting responsive RNA element TAR is necessary for activation of HIV-1 gene expression. We investigated the hypothesis that the essential uridine residue at position 23 in the bulge of TAR RNA is involved in intramolecular hydrogen bonding to stabilize an unique RNA structure required for recognition by Tat. Nucleotide substitutions in the two base pairs of the TAR stem directly above the essential trinucleotide bulge that maintain base pairing but change sequence prevent complex formation with Tat in vitro. Corresponding mutations tested in a trans-activation assay strongly affect the biological activity of TAR in vivo, suggesting an important role for these nucleotides in the Tat-TAR interaction. On the basis of these data, a model is proposed which implicates uridine 23 in a stable tertiary interaction with the GC pair directly above the bulge. This interaction would cause widening of the major groove of the RNA, thereby exposing its hydrogen-bonding surfaces for possible interaction with Tat. The model also predicts a gap between uridine 23 and the first base pair in the stem above, which would require one or more unpaired nucleotides to close, but does not predict any other role for such nucleotides. In accordance with this prediction, synthetic propyl phosphate linkers of equivalent length to 1 or 2 nucleotides, were found to be fully acceptable substitutes in the bulge above uridine 23, demonstrating that neither the bases nor the ribose moieties at these positions are implicated in the recognition of TAR RNA by Tat. 相似文献