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1.
The RNA world hypothesis states that the early evolution of life went through a stage where RNA served as genome and as catalyst. The replication of RNA world organisms would have been facilitated by ribozymes that catalyze RNA polymerization. To recapitulate an RNA world in the laboratory, a series of RNA polymerase ribozymes was developed previously. However, these ribozymes have a polymerization efficiency that is too low for self-replication, and the most efficient ribozymes prefer one specific template sequence. The limiting factor for polymerization efficiency is the weak sequence-independent binding to its primer/template substrate. Most of the known polymerase ribozymes bind an RNA heptanucleotide to form the P2 duplex on the ribozyme. By modifying this heptanucleotide, we were able to significantly increase polymerization efficiency. Truncations at the 3'-terminus of this heptanucleotide increased full-length primer extension by 10-fold, on a specific template sequence. In contrast, polymerization on several different template sequences was improved dramatically by replacing the RNA heptanucleotide with DNA oligomers containing randomized sequences of 15 nt. The presence of G and T in the random sequences was sufficient for this effect, with an optimal composition of 60% G and 40% T. Our results indicate that these DNA sequences function by establishing many weak and nonspecific base-pairing interactions to the single-stranded portion of the template. Such low-specificity interactions could have had important functions in an RNA world. 相似文献
2.
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
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Piccirilli JA Koldobskaya Y 《Philosophical transactions of the Royal Society of London. Series B, Biological sciences》2011,366(1580):2918-2928
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
4.
Westhof E 《Journal of molecular recognition : JMR》2007,20(1):1-3
A new crystal structure of the hammerhead ribozyme demonstrates the influence of peripheral tertiary contacts on the local conformations around the active site. This structure resolves many conflicting results obtained on reduced systems. 相似文献
5.
Masatora Fukuda Kei Kurihara Shota Yamaguchi Yui Oyama Masanobu Deshimaru 《RNA (New York, N.Y.)》2014,20(3):392-405
Adenosine-to-inosine (A-to-I) RNA editing is an endogenous regulatory mechanism involved in various biological processes. Site-specific, editing-state–dependent degradation of target RNA may be a powerful tool both for analyzing the mechanism of RNA editing and for regulating biological processes. Previously, we designed an artificial hammerhead ribozyme (HHR) for selective, site-specific RNA cleavage dependent on the A-to-I RNA editing state. In the present work, we developed an improved strategy for constructing a trans-acting HHR that specifically cleaves target editing sites in the adenosine but not the inosine state. Specificity for unedited sites was achieved by utilizing a sequence encoding the intrinsic cleavage specificity of a natural HHR. We used in vitro selection methods in an HHR library to select for an extended HHR containing a tertiary stabilization motif that facilitates HHR folding into an active conformation. By using this method, we successfully constructed highly active HHRs with unedited-specific cleavage. Moreover, using HHR cleavage followed by direct sequencing, we demonstrated that this ribozyme could cleave serotonin 2C receptor (HTR2C) mRNA extracted from mouse brain, depending on the site-specific editing state. This unedited-specific cleavage also enabled us to analyze the effect of editing state at the E and C sites on editing at other sites by using direct sequencing for the simultaneous quantification of the editing ratio at multiple sites. Our approach has the potential to elucidate the mechanism underlying the interdependencies of different editing states in substrate RNA with multiple editing sites. 相似文献
6.
Novel ribozymes produced by in vitro selection techniques provide insights into the possible mechanisms of protein synthesis evolution. The availability of such ribozymes also paves the way for experiments to explore the evolution of RNA–protein enzymes. 相似文献
7.
The hairpin ribozyme 总被引:1,自引:0,他引:1
Ferré-D'Amaré AR 《Biopolymers》2004,73(1):71-78
The hairpin ribozyme is a naturally occurring RNA that catalyzes sequence-specific cleavage and ligation of RNA. It has been the subject of extensive biochemical and structural studies, perhaps the most detailed for any catalytic RNA to date. Comparison of the structures of its constituent domains free and fully assembled demonstrates that the RNA undergoes extensive structural rearrangement. This rearrangement results in a distortion of the substrate RNA that primes it for cleavage. This ribozyme is known to achieve catalysis employing exclusively RNA functional groups. Metal ions or other catalytic cofactors are not used. Current experimental evidence points to a combination of at least four mechanistic strategies by this RNA: (1) precise substrate orientation, (2) preferential transition state binding, (3) electrostatic catalysis, and (4) general acid base catalysis. 相似文献
8.
Amplification of an RNA template molecule was examined using the ligase ribozyme and its corresponding RNA substrates under alternating temperature conditions. Alternating temperatures enhanced the rate of the thermodynamically unfavorable dissociation of the annealed products into the two separate RNA templates, reminiscent of the polymerase chain reaction. Under these conditions, the RNA ligase ribozyme system was observed to amplify through a mainly cross-catalytic process, generating additional copies of the starting RNA template molecules. Thus, template-directed RNA ligation using the ribozyme under thermally fluctuating conditions will be an intriguing point to consider when explaining the primordial event of chemical evolution. 相似文献
9.
The hairpin ribozyme is a small catalytic RNA that accelerates reversible cleavage of a phosphodiester bond. Structural and mechanistic studies suggest that divalent metals stabilize the functional structure but do not participate directly in catalysis. Instead, two active site nucleobases, G8 and A38, appear to participate in catalytic chemistry. The features of A38 that are important for active site structure and chemistry were investigated by comparing cleavage and ligation reactions of ribozyme variants with A38 modifications. An abasic substitution of A38 reduced cleavage and ligation activity by 14,000-fold and 370,000-fold, respectively, highlighting the critical role of this nucleobase in ribozyme function. Cleavage and ligation activity of unmodified ribozymes increased with increasing pH, evidence that deprotonation of some functional group with an apparent pK(a) value near 6 is important for activity. The pH-dependent transition in activity shifted by several pH units in the basic direction when A38 was substituted with an abasic residue, or with nucleobase analogs with very high or low pK(a) values that are expected to retain the same protonation state throughout the experimental pH range. Certain exogenous nucleobases that share the amidine group of adenine restored activity to abasic ribozyme variants that lack A38. The pH dependence of chemical rescue reactions also changed according to the intrinsic basicity of the rescuing nucleobase, providing further evidence that the protonation state of the N1 position of purine analogs is important for rescue activity. These results are consistent with models of the hairpin ribozyme catalytic mechanism in which interactions with A38 provide electrostatic stabilization to the transition state. 相似文献
10.
Substitutional RNA editing plays a crucial role in the regulation of biological processes. Cleavage of target RNA that depends on the specific site of substitutional RNA editing is a useful tool for analyzing and regulating intracellular processes related to RNA editing. Hammerhead ribozymes have been utilized as small catalytic RNAs for cleaving target RNA at a specific site and may be used for RNA-editing-specific RNA cleavage. Here we reveal a design strategy for a hammerhead ribozyme that specifically recognizes adenosine to inosine (A-to-I) and cytosine to uracil (C-to-U) substitutional RNA-editing sites and cleaves target RNA. Because the hammerhead ribozyme cleaves one base upstream of the target-editing site, the base that pairs with the target-editing site was utilized for recognition. RNA-editing-specific ribozymes were designed such that the recognition base paired only with the edited base. These ribozymes showed A-to-I and C-to-U editing-specific cleavage activity against synthetic serotonin receptor 2C and apolipoprotein B mRNA fragments in vitro, respectively. Additionally, the ribozyme designed for recognizing A-to-I RNA editing at the Q/R site on filamin A (FLNA) showed editing-specific cleavage activity against physiologically edited FLNA mRNA extracted from cells. We demonstrated that our strategy is effective for cleaving target RNA in an editing-dependent manner. The data in this study provided an experimental basis for the RNA-editing-dependent degradation of specific target RNA in vivo. 相似文献
11.
Chauhan S Caliskan G Briber RM Perez-Salas U Rangan P Thirumalai D Woodson SA 《Journal of molecular biology》2005,353(5):1199-1209
Large RNAs collapse into compact intermediates in the presence of counterions before folding to the native state. We previously found that collapse of a bacterial group I ribozyme correlates with the formation of helices within the ribozyme core, but occurs at Mg2+ concentrations too low to support stable tertiary structure and catalytic activity. Here, using small-angle X-ray scattering, we show that Mg2+-induced collapse is a cooperative folding transition that can be fit by a two-state model. The Mg2+ dependence of collapse is similar to the Mg2+ dependence of helix assembly measured by partial ribonuclease T1 digestion and of an unfolding transition measured by UV hypochromicity. The correspondence between multiple probes of RNA structure further supports a two-state model. A mutation that disrupts tertiary contacts between the L9 tetraloop and its helical receptor destabilized the compact state by 0.8 kcal/mol, while mutations in the central triplex were less destabilizing. These results show that native tertiary interactions stabilize the compact folding intermediates under conditions in which the RNA backbone remains accessible to solvent. 相似文献
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13.
A helical twist-induced conformational switch activates cleavage in the hammerhead ribozyme 总被引:6,自引:0,他引:6
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
14.
The catalytic activity of the hammerhead ribozyme is limited by its ability to fold into the native tertiary structure. Analysis of folding has been hampered by a lack of assays that can independently monitor the environment of nucleobases throughout the ribozyme-substrate complex in real time. Here, we report the development and application of a new folding assay in which we use pyrrolo-cytosine (pyC) fluorescence to (1) probe active-site formation, (2) examine the ability of peripheral ribozyme domains to support native folding, (3) identify a pH-dependent conformational change within the ribozyme, and (4) explore its influence on the equilibrium between the folded and unfolded core of the hammerhead ribozyme. We conclude that the natural ribozyme folds in two distinct noncooperative steps and the pH-dependent correlation between core folding and activity is linked to formation of the G8-C3 base pair. 相似文献
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Determination of quantitative thermodynamic and kinetic frameworks for ribozymes derived from the Azoarcus group I intron and comparisons to their well-studied analogs from the Tetrahymena group I intron reveal similarities and differences between these RNAs. The guanosine (G) substrate binds to the Azoarcus and Tetrahymena ribozymes with similar equilibrium binding constants and similar very slow association rate constants. These and additional literature observations support a model in which the free ribozyme is not conformationally competent to bind G and in which the probability of assuming the binding-competent state is determined by tertiary interactions of peripheral elements. As proposed previously, the slow binding of guanosine may play a role in the specificity of group I intron self-splicing, and slow binding may be used analogously in other biological processes. The internal equilibrium between ribozyme-bound substrates and products is similar for these ribozymes, but the Azoarcus ribozyme does not display the coupling in the binding of substrates that is observed with the Tetrahymena ribozyme, suggesting that local preorganization of the active site and rearrangements within the active site upon substrate binding are different for these ribozymes. Our results also confirm the much greater tertiary binding energy of the 5′-splice site analog with the Azoarcus ribozyme, binding energy that presumably compensates for the fewer base-pairing interactions to allow the 5′-exon intermediate in self splicing to remain bound subsequent to 5′-exon cleavage and prior to exon ligation. Most generally, these frameworks provide a foundation for design and interpretation of experiments investigating fundamental properties of these and other structured RNAs. 相似文献
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19.
Polyamines are abundant metabolites that directly influence gene expression. Although the role of polyamines in DNA condensation is well known, their role in RNA folding is less understood. Non-denaturing gel electrophoresis was used to monitor the equilibrium folding transitions of the Tetrahymena ribozyme in the presence of polyamines. All of the polyamines tested induce near-native structures that readily convert to the native conformation in Mg(2+). The stability of the folded structure increases with the charge of the polyamine and decreases with the size of the polyamine. When the counterion excluded volume becomes large, the transition to the native state does not go to completion even under favorable folding conditions. Brownian dynamics simulations of a model polyelectrolyte suggest that the kinetics of counterion-mediated collapse and the dimensions of the collapsed RNA chains depend on the structure of the counterion. The results are consistent with delocalized condensation of polyamines around the RNA. However, the effective charge of the counterions is lowered by their excluded volume. The stability of the folded RNA is enhanced when the spacing between amino groups matches the distance between adjacent phosphate groups. These results show how changes in intracellular polyamine concentrations could alter RNA folding pathways. 相似文献
20.
The effects of various metal ions on cleavage activity and global folding have been studied in the extended Schistosoma hammerhead ribozyme. Fluorescence resonance energy transfer was used to probe global folding as a function of various monovalent and divalent metal ions in this ribozyme. The divalent metals ions Ca2+, Mg2+, Mn2+, and Sr2+ have a relatively small variation (less than sixfold) in their ability to globally fold the hammerhead ribozyme, which contrasts with the very large difference (>10,000-fold) in apparent rate constants for cleavage for these divalent metal ions in single-turnover kinetic experiments. There is still a very large range (>4600-fold) in the apparent rate constants for cleavage for these divalent metal ions measured in high salt (2 M NaCl) conditions where the ribozyme is globally folded. These results demonstrate that the identity of the divalent metal ion has little effect on global folding of the Schistosoma hammerhead ribozyme, whereas it has a very large effect on the cleavage kinetics. Mechanisms by which the identity of the divalent metal ion can have such a large effect on cleavage activity in the Schistosoma hammerhead ribozyme are discussed. 相似文献