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1.
The tolerance to exchanges of the Watson Crick base pair in the hammerhead ribozyme core is determined by surrounding elements
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Tertiary interacting elements are important features of functional RNA molecules, for example, in all small nucleolytic ribozymes. The recent crystal structure of a tertiary stabilized type I hammerhead ribozyme revealed a conventional Watson-Crick base pair in the catalytic core, formed between nucleotides C3 and G8. We show that any Watson-Crick base pair between these positions retains cleavage competence in two type III ribozymes. In the Arabidopsis thaliana sequence, only moderate differences in cleavage rates are observed for the different base pairs, while the peach latent mosaic viroid (PLMVd) ribozyme exhibits a preference for a pyrimidine at position 3 and a purine at position 8. To understand these differences, we created a series of chimeric ribozymes in which we swapped sequence elements that surround the catalytic core. The kinetic characterization of the resulting ribozymes revealed that the tertiary interacting loop sequences of the PLMVd ribozyme are sufficient to induce the preference for Y3-R8 base pairs in the A. thaliana hammerhead ribozyme. In contrast to this, only when the entire stem-loops I and II of the A. thaliana sequences are grafted on the PLMVd ribozyme is any Watson-Crick base pair similarly tolerated. The data provide evidence for a complex interplay of secondary and tertiary structure elements that lead, mediated by long-range effects, to an individual modulation of the local structure in the catalytic core of different hammerhead ribozymes. 相似文献
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3.
A long-range pseudoknot is required for activity of the Neurospora VS ribozyme. 总被引:2,自引:0,他引:2
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Four small RNA self-cleaving domains, the hammerhead, hairpin, hepatitis delta virus and Neurospora VS ribozymes, have been identified previously in naturally occurring RNAs. The secondary structures of these ribozymes are reasonably well understood, but little is known about long-range interactions that form the catalytically active tertiary conformations. Our previous work, which identified several secondary structure elements of the VS ribozyme, also showed that many additional bases were protected by magnesium-dependent interactions, implying that several tertiary contacts remained to be identified. Here we have used site-directed mutagenesis and chemical modification to characterize the first long-range interaction identified in VS RNA. This interaction contains a 3 bp pseudoknot helix that is required for tertiary folding and self-cleavage activity of the VS ribozyme. 相似文献
4.
A three-nucleotide helix I is sufficient for full activity of a hammerhead ribozyme: advantages of an asymmetric design. 总被引:4,自引:3,他引:1
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Trans-cleaving hammerhead ribozymes with long target-specific antisense sequences flanking the catalytic domain share some features with conventional antisense RNA and are therefore termed 'catalytic antisense RNAs'. Sequences 5' to the catalytic domain form helix I and sequences 3' to it form helix III when complexed with the target RNA. A catalytic antisense RNA of more than 400 nucleotides, and specific for the human immunodeficiency virus type 1 (HIV-1), was systematically truncated within the arm that constituted originally a helix I of 128 base pairs. The resulting ribozymes formed helices I of 13, 8, 5, 3, 2, 1 and 0 nucleotides, respectively, and a helix III of about 280 nucleotides. When their in vitro cleavage activity was compared with the original catalytic antisense RNA, it was found that a helix I of as little as three nucleotides was sufficient for full endonucleolytic activity. The catalytically active constructs inhibited HIV-1 replication about four-fold more effectively than the inactive ones when tested in human cells. A conventional hammerhead ribozyme having helices of just 8 nucleotides on either side failed to cleave the target RNA in vitro when tested under the conditions for catalytic antisense RNA. Cleavage activity could only be detected after heat-treatment of the ribozyme substrate mixture which indicates that hammerhead ribozymes with short arms do not associate as efficiently to the target RNA as catalytic antisense RNA. The requirement of just a three-nucleotide helix I allows simple PCR-based generation strategies for asymmetric hammerhead ribozymes. Advantages of an asymmetric design will be discussed. 相似文献
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6.
Fast formation of the P3-P7 pseudoknot: a strategy for efficient folding of the catalytically active ribozyme 总被引:2,自引:0,他引:2
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Formation of the P3-P7 pseudoknot structure, the core of group I ribozymes, requires long-range base pairing. Study of the Tetrahymena ribozyme appreciates the hierarchical folding of the large, multidomain RNA, in which the P3-P7 core folds significantly slower than do the other domains. Here we explored the formation of the P3-P7 pseudoknot of the Candida ribozyme that has been reported to concertedly fold to the catalytically active structure with a rate constant of 2 min(-1). We demonstrate that pseudoknot formation occurs during the rapid ribozyme compaction, coincident with formation of many tertiary interactions of the ribozyme. A low physiological concentration of magnesium (1.5 mM) is sufficient to fully support the pseudoknot formation. The presence of nonnative intermediates containing an unfolded P3-P7 region is evident. However, catalysis-based analysis shows these nonnative intermediates are stable and fail to convert to the catalytically active structure, suggesting that rapid pseudoknot formation is essential for folding of the active ribozyme. Interestingly, RNAstructure predicts no stable Alt P3 structure for the Candida ribozyme, but two stable Alt P3s for the Tetrahymena ribozyme, explaining the dramatic difference in folding of the P3-P7 core of these two ribozymes. We propose that rapid formation of the P3-P7 pseudoknot represents a folding strategy ensuring efficient production of the catalytically active structure of group I ribozymes, which sheds new light on the mechanism of effective ribozyme folding in vivo. 相似文献
7.
Site-specific modification of functional groups in genomic hepatitis delta virus (HDV) ribozyme. 总被引:3,自引:0,他引:3
Fumiko Nishikawa Miho Shirai Satoshi Nishikawa 《European journal of biochemistry》2002,269(23):5792-5803
Human hepatitis delta (HDV) ribozyme is one of small ribozymes, such as hammerhead and hairpin ribozymes, etc. Its secondary structure shows pseudoknot structure composed of four stems (I to IV) and three single-stranded regions (SSrA, -B and -C). The 3D structure of 3'-cleaved product of genomic HDV ribozyme provided extensive information about tertiary hydrogen bonding interactions between nucleotide bases, phosphate oxygens and 2'OHs including new stem structure P1.1. To analyze the role of these hydrogen bond networks in the catalytic reaction, site-specific atomic-level modifications (such as deoxynucleotides, deoxyribosyl-2-aminopurine, deoxyribosylpurine, 7-deaza-ribonucleotide and inosine) were incorporated in the smallest trans-acting HDV ribozyme (47-mer). Kinetic analysis of these ribozyme variants demonstrated the importance of the two W-C base pairs of P1.1 for cleavage; in addition, the results suggest that all hydrogen bond interactions detected in the crystal structure involving 2'-OH and N7 atoms are present in the active ribozyme structure. In most of the variants, the relative reduction in kobs caused by substitution of the 2'-OH group correlated with the number of hydrogen bonds affected by the substitution. However G74 and C75 may have more than one hydrogen bond involving the 2'-OH in both the trans- and cis-acting HDV ribozyme. Moreover, in variants in which N7 was deleted, kobs was reduced 5- to 15-fold, it may suggest that N7 assists in coordinating Mg2+ ions or water molecules which bind with weak affinity in the active structure. 相似文献
8.
Chimeric DNA-RNA hammerhead ribozymes have enhanced in vitro catalytic efficiency and increased stability in vivo. 总被引:13,自引:12,他引:1
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Subsequent to the discovery that RNA can have site specific cleavage activity, there has been a great deal of interest in the design and testing of trans-acting catalytic RNAs as both surrogate genetic tools and as therapeutic agents. We have been developing catalytic RNAs or ribozymes with target specificity for HIV-1 RNA and have been exploring chemical synthesis as one method for their production. To this end, we have chemically synthesized and experimentally analyzed chimeric catalysts consisting of DNA in the non-enzymatic portions, and RNA in the enzymatic core of hammerhead type ribozymes. Substitutions of DNA for RNA in the various stems of a hammerhead ribozyme have been analyzed in vitro for kinetic efficiency. One of the chimeric ribozymes used in this study, which harbors 24 bases of DNA capable of base-pairing interactions with an HIV-1 gag target, but maintains RNA in the catalytic center and in stem-loop II, has a sixfold greater kcat value than the all RNA counterpart. This increased activity appears to be the direct result of enhanced product dissociation. Interestingly, a chimeric ribozyme in which stem-loop II (which divides the catalytic core) is comprised of DNA, exhibited a marked reduction in cleavage activity, suggesting that DNA in this region of the ribozyme can impart a negative effect on the catalytic function of the ribozyme. DNA-RNA chimeric ribozymes transfected by cationic liposomes into human T-lymphocytes are more stable than their all-RNA counterparts. Enhanced catalytic turnover and stability in the absence of a significant effect on Km make chimeric ribozymes favorable candidates for therapeutic agents. 相似文献
9.
Synthetic regulatory devices are key components for the development of complex biological systems and the reprogramming of cellular functions and networks. Here we describe the selection of tetracycline inducible hammerhead ribozymes. A tetracycline aptamer was fused to the full-length hammerhead ribozyme via a variable linker region. 11 rounds of in vitro selection were applied to isolate linker sequences that mediate tetracycline dependent hammerhead cleavage. We identified allosteric ribozymes that cleave in the presence of 1 μM tetracycline as fast as the full-length ribozyme whereas cleavage is inhibited up to 333-fold in the absence of tetracycline. Reporter gene assays indicate that the allosteric ribozymes can be employed to control gene expression in yeast. 相似文献
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11.
The effect of structure in a long target RNA on ribozyme cleavage efficiency. 总被引:2,自引:0,他引:2
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Inhibition of gene expression by catalytic RNA (ribozymes) requires that ribozymes efficiently cleave specific sites within large target RNAs. However, the cleavage of long target RNAs by ribozymes is much less efficient than cleavage of short oligonucleotide substrates because of higher order structure in the long target RNA. To further study the effects of long target RNA structure on ribozyme cleavage efficiency, we determined the accessibility of seven hammerhead ribozyme cleavage sites in a target RNA that contained human immunodeficiency virus type 1 (HIV-1) vif - vpr . The base pairing-availability of individual nucleotides at each cleavage site was then assessed by chemical modification mapping. The ability of hammerhead ribozymes to cleave the long target RNA was most strongly correlated with the availability of nucleotides near the cleavage site for base pairing with the ribozyme. Moreover, the accessibility of the seven hammerhead ribozyme cleavage sites in the long target RNA varied by up to 400-fold but was directly determined by the availability of cleavage sites for base pairing with the ribozyme. It is therefore unlikely that steric interference affected hammerhead ribozyme cleavage. Chemical modification mapping of cleavage site structure may therefore provide a means to identify efficient hammerhead ribozyme cleavage sites in long target RNAs. 相似文献
12.
A new type of hammerhead ribozyme, with cleavage activity enhanced by oligonucleotides, was constructed. Stem II of the ribozyme was substituted with a non complementary loop (loop II). The modified ribozyme exhibited negligible cleavage of a target RNA; however, it was converted to an active molecule in the presence of oligonucleotides which were complementary to loop II. The oligonucleotide compensated for the disabled stem II by binding with the ribozyme. The induction of the cleavage activity was sequence-specific and the oligonucleotides containing a purine base as the 3'-dangling end were able to induce the cleavage activity of the ribozyme most efficiently. A photo-crosslinking experiment proved that a pseudo-half-knot structure was formed in the active molecule. The cleavage of two kinds of substrate RNAs with different sequences was controlled by the corresponding ribozymes activated by specific oligonucleotides. 相似文献
13.
William G. Scott Monika Martick Young-In Chi 《Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms》2009,1789(9-10):634-641
Since their discovery in the 1980s, it has gradually become apparent that there are several functional classes of naturally occurring ribozymes. These include ribozymes that mediate RNA splicing (the Group I and Group II introns, and possibly the RNA components of the spliceosome), RNA processing ribozymes (RNase P, which cleaves precursor tRNAs and other structural RNA precursors), the peptidyl transferase center of the ribosome, and small, self-cleaving genomic ribozymes (including the hammerhead, hairpin, HDV and VS ribozymes). The most recently discovered functional class of ribozymes include those that are embedded in the untranslated regions of mature mRNAs that regulate the gene's translational expression. These include the prokaryotic glmS ribozyme, a bacterial riboswitch, and a variant of the hammerhead ribozyme, which has been found embedded in mammalian mRNAs. With the discovery of a mammalian riboswitch ribozyme, the question of how an embedded hammerhead ribozyme's switching mechanism works becomes a compelling question. Recent structural results suggest several possibilities. 相似文献
14.
借助计算机软件分析 ,设计出能特异性切割HPV11型 6 4 4ntE2mRNA的核酶 (ribozyme) .遵循Symon′s锤头状核酶结构和GUX剪切位点原则 ,靶序列存在 32个这样的剪切位点 .通过计算机软件分析出核酶的最佳剪切位点 ,并对底物及核酶的二级结构进行预测及进行相应基因生物学功能和基因同源性分析 ,筛选出 2个锤头结构核酶 .针对这两位点设计的核酶分别命名为RZ2 777和RZ32 81.计算机分析显示 ,两核酶与底物切点两翼碱基形成锤头状结构 ,切点所在基因序列具有相对松弛的二级结构 ,位于该基因重要生物功能区内 ,是核酶的理想攻击区域 .通过基因库检索 ,在已知人类基因排除了与上述两核酶切点两翼碱基有基因同源性序列的可能性 .将两核酶用于体外剪切实验取得了良好的实验结果 ,认为借助计算机分析可帮助尽快从多个剪切位点选择出最适核酶 相似文献
15.
Nyholm T Andäng M Bandholtz A Maijgren C Persson B Hotchkiss G Fehniger TE Larsson S Ahrlund-Richter L 《Journal of biochemical and biophysical methods》2000,44(1-2):41-57
Dynamic interactions between hammerhead ribozymes and RNA substrates were measured using the surface plasmon resonance (SPR) technology. Two in vitro transcribed substrates (non-cleavable and cleavable) were immobilised on streptavidin-coated dextran matrices and subsequently challenged with non-related yeast tRNA or two hammerhead ribozymes, both of which had previously been shown to exhibit functional binding and cleavage of complementary target RNAs. The target-binding domain of one of the ribozymes was fully complementary to a 16-ribonucleotide stretch on the immobilised substrates, while the other ribozyme had a nine-ribonucleotide complementarity. The two ribozymes could readily be differentiated with regard to affinity. Cleavage could be measured, using the ribozyme with full target complementarity to the cleavable substrate. In contrast, the ribozyme with lower affinity lacked cleavage activity. We suggest that SPR will be useful for investigations of ribozyme-substrate interactions. 相似文献
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17.
trans-Cleaving hammerhead ribozyme variants were generated with mimicked non-conserved internal loop motifs derived from five structurally diverse natural cis-cleaving ribozymes. Most modified trans-cleaving variants showed enhanced single-turnover cleavage rates relative to minimal counterparts that lack tertiary interactions between internal loop motifs I and II, and relative to controls with sequence changes in loop I. The trans-cleaving ribozyme derived from the positive strand of peach latent mosaic viroid had the highest observed cleavage rate, suggesting a structurally optimized motif that facilitates rapid formation of the ribozyme catalytic center in a trans-reaction. 相似文献
18.
Effects of variations in length of hammerhead ribozyme antisense arms upon the cleavage of longer RNA substrates. 总被引:1,自引:0,他引:1
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M Sioud 《Nucleic acids research》1997,25(2):333-338
The efficacy of intracellular binding of hammerhead ribozyme to its cleavage site in target RNA is a major requirement for its use as a therapeutic agent. Such efficacy can be influenced by several factors, such as the length of the ribozyme antisense arms and mRNA secondary structures. Analysis of various IL-2 hammerhead ribozymes having different antisense arms but directed to the same site predicts that the hammerhead ribozyme target site is present within a double-stranded region that is flanked by single-stranded loops. Extension of the low cleaving hammerhead ribozyme antisense arms by nucleotides that base pair with the single-stranded regions facilitated the hammerhead ribozyme binding to longer RNA substrates (e.g. mRNA). In addition, a correlation between the in vitro and intracellular results was also found. Thus, the present study would facilitate the design of hammerhead ribozymes directed against higher order structured sites. Further, it emphasises the importance of detailed structural investigations of hammerhead ribozyme full-length target RNAs. 相似文献
19.
Komatsu Y Yamashita S Kazama N Nobuoka K Ohtsuka E 《Journal of molecular biology》2000,299(5):1231-1243
A hairpin loop and an oligonucleotide bound to the loop form one-half of the pseudoknot structure. We have designed an allosteric hammerhead ribozyme, which is activated by the introduction of this motif by using a short complementary oligonucleotide as a cofactor. Stem II of the hammerhead ribozyme was substituted with a non-self-complementary loop sequence (loop II) to abolish the cleavage activity. The new ribozyme had almost no cleavage activity of the target RNA. However, it exhibited the cleavage activity in the presence of a cofactor oligoribonucleotide, which is complementary to loop II of the ribozyme. The activity is assumed to be derived from the formation of a pseudo-stem structure between the cofactor oligonucleotide and loop II. The structure including the loop may be similar to the pseudo-half-knot structure. The activation efficiencies of the cofactor oligonucleotides were decreased as the lengths of the oligonucleotides increased, and the ribozyme with a longer loop II was more active than that with a short loop II. Oligoribonucleotides with 3'-dangling purine bases served as efficient cofactors of the ribozyme, and a 2'-O-methyloligonucleotide enhanced the cleavage activity of the ribozyme most efficiently, by as much as about 750-fold as compared with that in the absence of the oligonucleotide. Cofactor oligonucleotides with a cytidine base at the 3'-end also activated a ribozyme with the G10.1.G11.1 mutation, which eliminates the cleavage activity in the wild-type. The binding sites of the oligonucleotide were identified by photo-crosslinking experiments and were found to be the predicted sites in the loop. This is the first report of a design aimed at positively controlling the activity of ribozymes by employing a structural motif. This method can be applied to control the activities of other functional RNAs with hairpin loops. 相似文献
20.
借助计算机软件分析,设计出能特异性切割HPV11型644nt型644ntE2mNA的核酶。遵循Symons锤头状核酶结构和GUX剪切位点原则,靶序列存在32个剪切位点,通过计算机软件分析核酶的最佳剪切位点,并对底物及核酶的二级结构进行预测及进行相应基因生物学功能和基因同源性分析,筛选出2个锤头结构核酶。针对这两位点设计的核酶分别命名为RZ277和RZ3281。计算机分析显示,两核酶与底物切点两翼碱基形成锤头状结构,切点所在基因序列具有相对松驰的二级结构,位于该基因重要生物功能区内,是核酶的理想攻击区域,通过基因库检索,在已知人类基因中排除了与上述两核酶切点两翼碱基有基因同源性序列的可能性。并非所有的GUX位点(X:C、U、A)或CUX均可作为核酶的最佳剪切切割反应,为下一步将核酶用于细胞内和体内试验打下基础。 相似文献