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1.
Kim KS  Choi WH  Choi BR  Oh S  Yea SS  Yoon MY  Kim DE 《FEBS letters》2007,581(21):4065-4072
Self-replication process of the RNA ligase ribozyme molecules was investigated by using the modified RNA ligase ribozyme under alternating temperature condition that enhances turnover rate of the RNA ligation reaction. In our experiment, the RNA ligase ribozyme system mainly undergoes a cross-catalytic replication process, in which two ribozymes catalyze each other's synthesis from a total of four RNA substrates under alternating temperature condition, resulting in time-dependent accumulation of additional copies of the starting ribozymes in a reaction mixture. The present study demonstrates that cross-catalytic replication in nucleic acids system can be efficiently devised under the alternating temperature condition.  相似文献   

2.
A ligase ribozyme that accelerates the ligation reaction with an oligonucleotide under low pH conditions was identified by in vitro adaptation in a previous study. We examined the effects of further rounds of evolution to isolate a more active ribozyme. The ribozyme, which was obtained after four rounds of evolution, was randomly mutated, and the resultant RNA library was subjected to in vitro selection at low pH. One ribozyme isolated from the pool was found to react 8,000 times faster than the original b1 ribozyme at pH 4. The reaction rate of the isolated ribozyme was enhanced at various pH values, and its pH dependence was less than that of the original ribozyme or the ribozyme selected with four rounds of evolution. The reaction rate of the isolated ribozyme was reduced in the presence of 3' primer, the sequence of which is complementary to the 3' primer-binding site of the ligase ribozyme. This inhibition induced by the primer oligonucleotide binding to the ribozyme 3' region implies that the 3' region plays a role in the ligation reaction of the ribozyme.  相似文献   

3.
Glasner ME  Yen CC  Ekland EH  Bartel DP 《Biochemistry》2000,39(50):15556-15562
In support of the idea that certain RNA molecules might be able to catalyze RNA replication, a ribozyme was previously generated that synthesizes short segments of RNA in a reaction modeled after that of proteinaceous RNA polymerases. Here, we describe substrate recognition by this polymerase ribozyme. Altering base or sugar moieties of the nucleoside triphosphate only moderately affects its utilization, provided that the alterations do not disrupt Watson-Crick pairing to the template. Correctly paired nucleotides have both a lower K(m) and a higher k(cat), suggesting that differential binding and orientation each play roles in discriminating matched from mismatched nucleotides. Binding of the pyrophosphate leaving group appears weak, as evidenced by a very inefficient pyrophosphate-exchange reaction, the reverse of the primer-extension reaction. Indeed, substitutions at the gamma-phosphate can be tolerated, although poorly. Thio substitutions of oxygen atoms at the reactive phosphate exert effects similar to those seen with cellular polymerases, leaving open the possibility of an active site analogous to those of protein enzymes. The polymerase ribozyme, derived from an efficient RNA ligase ribozyme, can achieve the very fast k(cat) of the parent ribozyme when the substrate of the polymerase (GTP) is replaced by an extended substrate (pppGGA), in which the GA dinucleotide extension corresponds to the second and third nucleotides of the ligase. This suggests that the GA dinucleotide, which had been deleted when converting the ligase into a polymerase, plays an important role in orienting the 5'-terminal nucleoside. Polymerase constructs that restore this missing orientation function should achieve much more efficient and perhaps more accurate RNA polymerization.  相似文献   

4.
A novel ligase ribozyme was in vitro selected from a random-sequence RNA library including N(6)-aminohexyl-modified adenine residues in place of natural adenine residues. This ribozyme mediated the formation of a phosphodiester bond with a DNA oligonucleotide through condensation with a 5'-triphosphate moiety on the ribozyme. Among the clones isolated from this selection, one was shown to accelerate ligation about 250-fold compared to the original random-sequence RNA library. Almost no rate acceleration was observed when the N(6)-aminohexyl-groups on adenine residues were omitted. Furthermore, ligation was dependent on the presence of a template DNA oligonucleotide that bridged the two strands.  相似文献   

5.
The class I ligase was among the first ribozymes to have been isolated from random sequences and represents the catalytic core of several RNA-directed RNA polymerase ribozymes. The ligase is also notable for its catalytic efficiency and structural complexity. Here, we report an improved version of this ribozyme, arising from selection that targeted the kinetics of the chemical step. Compared with the parent ribozyme, the improved ligase achieves a modest increase in rate enhancement under the selective conditions and shows a sharp reduction in [Mg2+] dependence. Analysis of the sequences and kinetics of successful clones suggests which mutations play the greatest part in these improvements. Moreover, backbone and nucleobase interference maps of the parent and improved ligase ribozymes complement the newly solved crystal structure of the improved ligase to identify the functionally significant interactions underlying the catalytic ability and structural complexity of the ligase ribozyme.  相似文献   

6.
The three-dimensional architecture of the class I ligase ribozyme   总被引:2,自引:0,他引:2  
The class I ligase ribozyme catalyzes a Mg(++)-dependent RNA-ligation reaction that is chemically analogous to a single step of RNA polymerization. Indeed, this ribozyme constitutes the catalytic domain of an accurate and general RNA polymerase ribozyme. The ligation reaction is also very rapid in both single- and multiple-turnover contexts and thus is informative for the study of RNA catalysis as well as RNA self-replication. Here we report the initial characterization of the three-dimensional architecture of the ligase. When the ligase folds, several segments become protected from hydroxyl-radical cleavage, indicating that the RNA adopts a compact tertiary structure. Ribozyme folding was largely, though not completely, Mg(++) dependent, with a K(1/2[Mg]) < 1 mM, and was observed over a broad temperature range (20 degrees C -50 degrees C). The hydroxyl-radical mapping, together with comparative sequence analyses and analogy to a region within 23S ribosomal RNA, were used to generate a three-dimensional model of the ribozyme. The predictive value of the model was tested and supported by a photo-cross-linking experiment.  相似文献   

7.
A main unsolved problem in the RNA World scenario for the origin of life is how a template-dependent RNA polymerase ribozyme emerged from short RNA oligomers obtained by random polymerization on mineral surfaces. A number of computational studies have shown that the structural repertoire yielded by that process is dominated by topologically simple structures, notably hairpin-like ones. A fraction of these could display RNA ligase activity and catalyze the assembly of larger, eventually functional RNA molecules retaining their previous modular structure: molecular complexity increases but template replication is absent. This allows us to build up a stepwise model of ligation-based, modular evolution that could pave the way to the emergence of a ribozyme with RNA replicase activity, step at which information-driven Darwinian evolution would be triggered.  相似文献   

8.
Circular hammerhead ribozymes were synthesized from linear oligoribonucleotides using T4 RNA ligase. Some of the precursors could not be efficiently circularized under standard conditions. For these molecules, the use of a DNA template allowed their efficient circularization. The template was designed to prevent the precursor from folding into an unsuitable structure. The template allowed circular ribozymes as small as 15 nucleotides in length to be efficiently synthesized at concentrations as high as 50 microM in the ligation reaction. The circular products retained their biological activity.  相似文献   

9.
The YFL ribozyme is an artificial ligase ribozyme isolated by a ‘design and selection’ strategy, in which a modular catalytic unit was generated on a rationally designed modular scaffold RNA. This ligase ribozyme has a versatile catalytic unit that accepts not only β-nicotinamide mononucleotide (β-NMN) but also inorganic pyrophosphate as leaving groups for template-dependent RNA ligation. Although this property is interesting from an evolutionary viewpoint regarding primitive RNA ligation/polymerization systems in the RNA world, structural analysis of the YFL ribozyme has not been continued due to apparent structural nonuniformity of its folded state. To elucidate the active structure of the YFL ribozyme, we performed in vitro evolution experiments to improve its folding ability. Biochemical and phylogenetic analyses of evolved variants indicated that the catalytic unit of the YFL ribozyme is compact and the 3′ single-stranded region of the parent YFL-1 ribozyme contributes to mask an element that could form an inactive structure.  相似文献   

10.
A ligase ribozyme accelerating a ligation reaction with oligonucleotide under a low-pH condition was selected by in vitro adaptation. A ribozyme active at pH 7 was randomly mutated, and the resultant RNA library was subjected to in vitro adaptation under a low-pH reaction condition. At pH 4, the adapted RNAs reacted with the oligonucleotide substrates about 200 times faster than the original ribozyme. When the ribozyme was cloned and sequenced, 10 of the 30 clones sequenced had identical sequences. The differences in sequence from the original ribozyme were found at four positions in the middle region and at the 3' end. A few sequential differences dominated the activity of the ribozyme under the extreme condition. The adapted ribozyme had one repeating sequence that was critical for the activity.  相似文献   

11.
In vitro selection can generate functional sequence variants of an RNA structural motif that are useful for comparative analysis. The technique is particularly valuable in cases where natural variation is unavailable or non-existent. We report the extension of this approach to a new extreme--the identification of a 112 nt ribozyme secondary structure imbedded within a 186 nt RNA. A pool of 10(14) variants of an RNA ligase ribozyme was generated using combinatorial chemical synthesis coupled with combinatorial enzymatic ligation such that 172 of the 186 relevant positions were partially mutagenized. Active variants of this pool were enriched using an in vitro selection scheme that retains the sequence variability at positions very close to the ligation junction. Ligases isolated after four rounds of selection catalyzed self-ligation up to 700 times faster than the starting sequence. Comparative analysis of the isolates indicated that when complexed with substrate RNAs the ligase forms a nested, double pseudo-knot secondary structure with seven stems and several important joining segments. Comparative analysis also suggested the identity of mutations that account for the increased activity of the selected ligase variants; designed constructs incorporating combinations of these changes were more active than any of the individual ligase isolates.  相似文献   

12.
邹正升  王升启 《病毒学报》1995,11(3):262-265
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13.
During an early step in the evolution of life, RNA served both as genome and as catalyst, according to the RNA world hypothesis. For self-replication, the RNA organisms must have contained an RNA that catalyzes RNA polymerization. As a first step toward recapitulating an RNA world in the laboratory, a polymerase ribozyme was generated previously by in vitro evolution and design. However, the efficiency of this ribozyme is about 100-fold too low for self-replication because of a low affinity of the ribozyme to its primer/template substrate. To improve the substrate interactions by colocalizing ribozyme and substrate on micelles, we attached hydrophobic anchors to both RNAs. We show here that the hydrophobic anchors led to aggregates with the expected size of the corresponding micelles. The micelle formation increased the polymerization yield of full-length products by 3- to 20-fold, depending on substrates and reaction conditions. With the best-characterized substrate, the improvement in polymerization efficiency was primarily due to reduced sequence-specific stalling on partially extended substrates. We discuss how, during the origin of life, micellar ribozyme aggregates could have acted as precursors to membrane-encapsulated life forms.  相似文献   

14.
All models of the RNA world era invoke the presence of ribozymes that can catalyse RNA polymerization. The class I ligase ribozyme selected in vitro 15 years ago from a pool of random RNA sequences catalyses formation of a 3',5'-phosphodiester linkage analogous to a single step of RNA polymerization. Recently, the three-dimensional structure of the ligase was solved in complex with U1A RNA-binding protein and independently in complex with an antibody fragment. The RNA adopts a tripod arrangement and appears to use a two-metal ion mechanism similar to protein polymerases. Here, we discuss structural implications for engineering a true polymerase ribozyme and describe the use of the antibody framework both as a portable chaperone for crystallization of other RNAs and as a platform for exploring steps in evolution from the RNA world to the RNA-protein world.  相似文献   

15.
The RNA world hypothesis states that the early evolution of life went through a stage in which RNA served both as genome and as catalyst. The central catalyst in an RNA world organism would have been a ribozyme that catalyzed RNA polymerization to facilitate self-replication. An RNA polymerase ribozyme was developed previously in the lab but it is not efficient enough for self-replication. The factor that limits its polymerization efficiency is its weak sequence-independent binding of the primer/template substrate. Here we tested whether RNA polymerization could be improved by a cationic arginine cofactor, to improve the interaction with the substrate. In an RNA world, amino acid-nucleic acid conjugates could have facilitated the emergence of the translation apparatus and the transition to an RNP world. We chose the amino acid arginine for our study because this is the amino acid most adept to interact with RNA. An arginine cofactor was positioned at ten different sites on the ribozyme, using conjugates of arginine with short DNA or RNA oligonucleotides. However, polymerization efficiency was not increased in any of the ten positions. In five of the ten positions the arginine reduced or modulated polymerization efficiency, which gives insight into the substrate-binding site on the ribozyme. These results suggest that the existing polymerase ribozyme is not well suited to using an arginine cofactor.  相似文献   

16.
The search is underway for a catalytic RNA molecule capable of self-replication. Finding such a ribozyme would lend crucial support to the RNA World hypothesis, which holds that very early life-forms relied on RNA for both replicating and storing genetic information. We previously reported an RNA polymerase isolated from a pool of variants of an existing RNA ligase ribozyme. Here we report eight additional ligase-derived polymerase ribozymes isolated from this pool. Because each of them is a new potential starting point for further in vitro evolution and engineering, together they substantially enrich the set of candidates from which an RNA replicase ribozyme might eventually emerge.  相似文献   

17.
We have captured the structure of a pre-catalytic conformational intermediate of the hammerhead ribozyme using a phosphodiester tether formed between I and Stem II. This phosphodiester tether appears to mimic interactions in the wild-type hammerhead RNA that enable switching between nuclease and ligase activities, both of which are required in the replicative cycles of the satellite RNA viruses from which the hammerhead ribozyme is derived. The structure of this conformational intermediate reveals how the attacking nucleophile is positioned prior to cleavage, and demonstrates how restricting the ability of Stem I to rotate about its helical axis, via interactions with Stem II, can inhibit cleavage. Analogous covalent crosslinking experiments have demonstrated that imposing such restrictions on interhelical movement can change the hammerhead ribozyme from a nuclease to a ligase. Taken together, these results permit us to suggest that switching between ligase and nuclease activity is determined by the helical orientation of Stem I relative to Stem II.  相似文献   

18.
The "RNA world" hypothesis rests on the assumption that RNA polymerase ribozymes can replicate RNA without the use of protein. In the laboratory, in vitro selection has been used to create primitive versions of such polymerases. The best variant to date is a ribozyme called B6.61 that can extend a RNA primer template by 20 nucleotides (nt). This polymerase has two domains: the recently crystallized Class I ligase core, responsible for phosphodiester bond formation, and the poorly characterized accessory domain that makes polymerization possible. Here we find that the accessory domain is specified by a 37-nt bulged stem-loop structure. The accessory domain is positioned by a tertiary interaction between the terminal AL4 loop of the accessory and the J3/4 triloop found within the ligase core. This docking interaction is associated with an unwinding of the A3 and A4 helixes that appear to facilitate the correct positioning of an essential 8-nt purine bulge found between the two helices. This, together with other constraints inferred from tethering the accessory domain to a range of sites on the ligase core, indicates that the accessory domain is draped over the vertex of the ligase core tripod structure. This geometry suggests how the purine bulge in the polymerase replaces the P2 helix in the Class I ligase with a new structure that may facilitate the stabilization of incoming nucleotide triphosphates.  相似文献   

19.
RNA-directed recombination can be used to catalyze a disproportionation reaction among small RNA substrates to create new combinations of sequences. But the accommodation of secondary and tertiary structural constraints in the substrates by recombinase ribozymes has not been explored. Here, we show that the Azoarcus group I intron can recombine oligoribonucleotides to construct class I ligase ribozymes, which are catalytically active upon synthesis. The substrate oligonucleotides, ranging in size from 58 to 104 nucleotides (nt), along with the 152-nt ligase ribozymes they reconstitute, can contain significant amounts of secondary structure. However, substrate recognition by the Azoarcus ribozyme depends on the existence of a single accessible CAU triplet for effective recombination. A biphasic temperature reaction profile was designed such that the sequential recombination/ligation events could take place in a thermocycler without human intervention. A temperature-dependent pH shift of the reaction buffer contributes to the success of the net reaction. When the substrate for the ligase ribozyme is introduced into the reaction mixture, as much as 11% can be observed being converted to product by the recombined ligase in the same reaction vessel. Recombination followed by ligation can also occur under isothermal conditions at 37 degrees C. Tertiary structure formation of the ligase upon construction can provide some protection from cleavage by the Azoarcus ribozyme when compared to the constituent substrates. These data suggest that RNA-directed recombination can, in fact, articulate complex ribozymes, and that there are logical rules that can guide the optimal placement of the CAU recognition sequence.  相似文献   

20.
Mg2? shares a distinctive relationship with RNA, playing important and specific roles in the folding and function of essentially all large RNAs. Here we use theory and experiment to evaluate Fe2? in the absence of free oxygen as a replacement for Mg2? in RNA folding and catalysis. We describe both quantum mechanical calculations and experiments that suggest that the roles of Mg2? in RNA folding and function can indeed be served by Fe2?. The results of quantum mechanical calculations show that the geometry of coordination of Fe2? by RNA phosphates is similar to that of Mg2?. Chemical footprinting experiments suggest that the conformation of the Tetrahymena thermophila Group I intron P4-P6 domain RNA is conserved between complexes with Fe2? or Mg2?. The catalytic activities of both the L1 ribozyme ligase, obtained previously by in vitro selection in the presence of Mg2?, and the hammerhead ribozyme are enhanced in the presence of Fe2? compared to Mg2?. All chemical footprinting and ribozyme assays in the presence of Fe2? were performed under anaerobic conditions. The primary motivation of this work is to understand RNA in plausible early earth conditions. Life originated during the early Archean Eon, characterized by a non-oxidative atmosphere and abundant soluble Fe2?. The combined biochemical and paleogeological data are consistent with a role for Fe2? in an RNA World. RNA and Fe2? could, in principle, support an array of RNA structures and catalytic functions more diverse than RNA with Mg2? alone.  相似文献   

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