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1.
酶是８０年代初期发现的具有自催化活性的ＲＮＡ片从核酸水平来破坏对人体不利的基因在人细胞内的表达,这是一种比较专一,且对人体危害相对较小的一种方式,经过改诉核酶不仅能起顺式作用,更重要的是也能以反式作用,这样就使得人们能够设计出针对ＲＮＡ的各种具有治疗作用的核酶,该文着重介绍具锤头型结构域和发夹型结构域的２类核酶的设计,及核酶作为治疗药物所要考虑的一些问题。 相似文献
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A critical event in the origin of life is thought to have been the emergence of an RNA molecule capable of replicating a primordial RNA "genome." Here we describe the evolution and engineering of an RNA polymerase ribozyme capable of synthesizing RNAs of up to 95 nucleotides in length. To overcome its sequence dependence, we recombined traits evolved separately in different ribozyme lineages. This yielded a more general polymerase ribozyme that was able to synthesize a wider spectrum of RNA sequences, as we demonstrate by the accurate synthesis of an enzymatically active RNA, a hammerhead endonuclease ribozyme. This recapitulates a central aspect of an RNA-based genetic system: the RNA-catalyzed synthesis of an active ribozyme from an RNA template. 相似文献
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Rupert PB Massey AP Sigurdsson ST Ferré-D'Amaré AR 《Science (New York, N.Y.)》2002,298(5597):1421-1424
The hairpin ribozyme catalyzes sequence-specific cleavage of RNA through transesterification of the scissile phosphate. Vanadate has previously been used as a transition state mimic of protein enzymes that catalyze the same reaction. Comparison of the 2.2 angstrom resolution structure of a vanadate-hairpin ribozyme complex with structures of precursor and product complexes reveals a rigid active site that makes more hydrogen bonds to the transition state than to the precursor or product. Because of the paucity of RNA functional groups capable of general acid-base or electrostatic catalysis, transition state stabilization is likely to be an important catalytic strategy for ribozymes. 相似文献
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[目的]利用核酶自我剪切功能使PRRSV的基因组获得1个精确的基因组3′末端,提高全长感染性cDNA克隆的病毒拯救效率。[方法]用3步PCR方法将获得含有丁型肝炎病毒核酶序列的片段并将其克隆到PRRSV全长cDNA克隆pAPRRS的poly（A）下游,再通过酶切,连接将牛生长激素多聚腺甘酸转录终止序列插入到核酶序列后,构建成全长感染性cDNA克隆pAPRRS-HB,新构建的克隆转染MARC-145细胞,72h后用间接免疫荧光检测病毒N蛋白和非结构蛋白2（nsp2）的表达,并用检测细胞上清中病毒的滴度。[结果]成功构建了含有核酶序列的PRRSV基因组全长感染性cDNA克隆pAPRRS-HB。新构建的全长感染性cDNA克隆能够较好地拯救出病毒,拯救效率比pAPRRS提高10倍左右。[结论]该结果为研究PRRSV基因结构与功能奠定了基础。 相似文献
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Kinetic characterization of ribonuclease-resistant 2'-modified hammerhead ribozymes 总被引：36，自引：0，他引：36
The incorporation of 2'-fluoro- and 2'-aminonucleotides into a hammerhead ribozyme was accomplished by automated chemical synthesis. The presence of 2'-fluorouridines, 2'-fluorocytidines, or 2'-aminouridines did not appreciably decrease catalytic efficiency. Incorporation of 2'-aminocytidines decreased ribozyme activity approximately by a factor of 20. The replacement of all adenosines with 2'-fluoroadenosines abolished catalysis in the presence of MgCl2 within the limits of detection, but some activity was retained in the presence of MnCl2. This effect on catalysis was localized to a specific group of adenines within the conserved single-stranded region of the ribozyme. The decrease in catalytic efficiency was caused by a decrease in the rate constant; the Michaelis constant was unaltered. The 2'-fluoro and 2'-amino modifications conferred resistance toward ribonuclease degradation. Ribozymes containing 2'-fluoro- or 2'-aminonucleotides at all uridine and cytidine positions were stabilized against degradation in rabbit serum by a factor of at least 10(3) compared to unmodified ribozyme. 相似文献
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Defining the inside and outside of a catalytic RNA molecule 总被引：42，自引：0，他引：42
Ribozymes are RNA molecules that catalyze biochemical reactions. Fe(II)-EDTA, a solvent-based reagent which cleaves both double- and single-stranded RNA, was used to investigate the structure of the Tetrahymena ribozyme. Regions of cleavage alternate with regions of substantial protection along the entire RNA molecule. In particular, most of the catalytic core shows greatly reduced cleavage. These data constitute experimental evidence that an RNA enzyme, like a protein enzyme, has an interior and an exterior. Determination of positions where the phosphodiester backbone of the RNA is on the inside or on the outside of the molecule provides major constraints for modeling the three-dimensional structure of the Tetrahymena ribozyme. This approach should be generally informative for structured RNA molecules. 相似文献
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The glmS ribozyme is the only natural catalytic RNA known to require a small-molecule activator for catalysis. This catalytic RNA functions as a riboswitch, with activator-dependent RNA cleavage regulating glmS messenger RNA expression. We report crystal structures of the glmS ribozyme in precleavage states that are unliganded or bound to the competitive inhibitor glucose-6-phosphate and in the postcleavage state. All structures superimpose closely, revealing a remarkably rigid RNA that contains a preformed active and coenzyme-binding site. Unlike other riboswitches, the glmS ribozyme binds its activator in an open, solvent-accessible pocket. Our structures suggest that the amine group of the glmS ribozyme-bound coenzyme performs general acid-base and electrostatic catalysis. 相似文献
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Many protein enzymes use general acid-base catalysis as a way to increase reaction rates. The amino acid histidine is optimized for this function because it has a pK(a) (where K(a) is the acid dissociation constant) near physiological pH. The RNA enzyme (ribozyme) from hepatitis delta virus catalyzes self-cleavage of a phosphodiester bond. Reactivity-pH profiles in monovalent or divalent cations, as well as distance to the leaving-group oxygen, implicate cytosine 75 (C75) of the ribozyme as the general acid and ribozyme-bound hydrated metal hydroxide as the general base in the self-cleavage reaction. Moreover, C75 has a pK(a) perturbed to neutrality, making it "histidine-like." Anticooperative interaction is observed between protonated C75 and a metal ion, which serves to modulate the pK(a) of C75. General acid-base catalysis expands the catalytic repertoire of RNA and may provide improved rate acceleration. 相似文献
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Stereochemistry of RNA cleavage by the Tetrahymena ribozyme and evidence that the chemical step is not rate-limiting 总被引：18，自引：0，他引：18
The intervening sequence of the ribosomal RNA precursor of Tetrahymena is a catalytic RNA molecule, or ribozyme. Acting as a sequence-specific endoribonuclease, it cleaves single-stranded RNA substrates with concomitant addition of guanosine. The chemistry of the reaction has now been studied by introduction of a single phosphorothioate in the substrate RNA at the cleavage site. Kinetic studies show no significant effect of this substitution on kcat (rate constant) or Km (Michaelis constant), providing evidence that some step other than the chemical step is rate-limiting. Product analysis reveals that the reaction proceeds with inversion of configuration at phosphorus, consistent with an in-line, SN2 (P) mechanism. Thus, the ribozyme reaction is in the same mechanistic category as the individual displacement reactions catalyzed by protein nucleotidyltransferases, phosphotransferases, and nucleases. 相似文献
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Zhuang X Bartley LE Babcock HP Russell R Ha T Herschlag D Chu S 《Science (New York, N.Y.)》2000,288(5473):2048-2051
Using fluorescence microscopy, we studied the catalysis by and folding of individual Tetrahymena thermophila ribozyme molecules. The dye-labeled and surface-immobilized ribozymes used were shown to be functionally indistinguishable from the unmodified free ribozyme in solution. A reversible local folding step in which a duplex docks and undocks from the ribozyme core was observed directly in single-molecule time trajectories, allowing the determination of the rate constants and characterization of the transition state. A rarely populated docked state, not measurable by ensemble methods, was observed. In the overall folding process, intermediate folding states and multiple folding pathways were observed. In addition to observing previously established folding pathways, a pathway with an observed folding rate constant of 1 per second was discovered. These results establish single-molecule fluorescence as a powerful tool for examining RNA folding. 相似文献
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简要讨论了近年来植物抗病毒基因工程的方法策略,主要有:植物自身的抗病毒基因策略、来源于病毒的抗性基因策略、干扰素、核酶等抗性策略;并分析了其存在问题及发展趋势。 相似文献
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Life originated, according to the RNA World hypothesis, from self-replicating ribozymes that catalyzed ligation of RNA fragments. We have solved the 2.6 angstrom crystal structure of a ligase ribozyme that catalyzes regiospecific formation of a 5' to 3' phosphodiester bond between the 5'-triphosphate and the 3'-hydroxyl termini of two RNA fragments. Invariant residues form tertiary contacts that stabilize a flexible stem of the ribozyme at the ligation site, where an essential magnesium ion coordinates three phosphates. The structure of the active site permits us to suggest how transition-state stabilization and a general base may catalyze the ligation reaction required for prebiotic RNA assembly. 相似文献
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为了建立传染性法氏囊病病毒反向遗传系统,应用PCR法在鸡传染性法氏囊病病毒基因组A、B两节段的5'末端和3'末端分别引入T7启动子和核酶HDV序列,然后分别将含有T7启动子和核酶HDV序列的A、B节段构建到载体PT-Pluc当中,构建感染性载体PT-mA和PT-mB.在脂质体介导下将PT-mA和PT-mB共转染经痘病毒vTF7-3(含T7 RNA聚合酶基因,能稳定表达T7 RNA聚合酶)预先感染1 h的vero细胞.细胞病变观测、间接免疫荧光试验、电镜观察和分子标记检测证实成功实现了鸡传染性法氏囊病病毒的遗传拯救. 相似文献
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We describe a single RNA sequence that can assume either of two ribozyme folds and catalyze the two respective reactions. The two ribozyme folds share no evolutionary history and are completely different, with no base pairs (and probably no hydrogen bonds) in common. Minor variants of this sequence are highly active for one or the other reaction, and can be accessed from prototype ribozymes through a series of neutral mutations. Thus, in the course of evolution, new RNA folds could arise from preexisting folds, without the need to carry inactive intermediate sequences. This raises the possibility that biological RNAs having no structural or functional similarity might share a common ancestry. Furthermore, functional and structural divergence might, in some cases, precede rather than follow gene duplication. 相似文献
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RNA as an RNA polymerase: net elongation of an RNA primer catalyzed by the Tetrahymena ribozyme 总被引：7，自引：0，他引：7
A catalytic RNA (ribozyme) derived from an intervening sequence (IVS) RNA of Tetrahymena thermophila will catalyze an RNA polymerization reaction in which pentacytidylic acid (C5) is extended by the successive addition of mononucleotides derived from a guanylyl-(3',5')-nucleotide (GpN). Cytidines or uridines are added to C5 to generate chain lengths of 10 to 11 nucleotides, with longer products being generated at greatly reduced efficiency. The reaction is analogous to that catalyzed by a replicase with C5 acting as the primer, GpNs as the nucleoside triphosphates, and a sequence in the ribozyme providing a template. The demonstration that an RNA enzyme can catalyze net elongation of an RNA primer supports theories of prebiotic RNA self-replication. 相似文献
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【目的】建立高效鸡传染性法氏囊病病毒（IBDV）拯救平台，为深入研究该病毒基因组的结构与功能奠定基础。【方法】在IBDV Gt株全基因组中引入分子标签（A节段：EcoR V酶切位点；B节段：PstI酶切位点）。在基因组两端分别引入锤头状核酶结构（HamRz）和丁肝病毒核酶结构（HdvRz)。将带有分子标签和核酶结构的IBDV基因组插入载体pCAGGS的β肌动蛋白启动子下游，构建IBDV感染性克隆pCAGGmGtAHRT和pCAGGmGtBHRT，LipofectamineTM2000介导共转染DFI细胞，进行病毒拯救研究。 【结果】 构建的IBDV感染性克隆可在DFI、Vero/E6、Vero/P12等3种细胞上高效拯救出病毒。RT-PCR、间接免疫荧光、电镜等均检测到了拯救的IBDV，且存在分子标签。拯救毒在CEF上能产生特有的砂砾状CPE，且TCID50与亲本毒差异不显著，具有相似的生物学特征。【结论】构建的RNA聚合酶ⅢBDV拯救系统高效、稳定、简便、经济，且具有良好的细胞普适性。 相似文献