New catalytic structures from an existing ribozyme

Curtis, Edward A.; Bartel, David P.

doi:10.1038/nsmb1003

Cited by 62 publications

(70 citation statements)

References 38 publications

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“…However, it should not be forgotten that a famously simple ribozyme, the hammerhead ribozyme, which catalyzes the cleavage/ligation of RNA chains, contains a core sequence only z20-30 nt, and the ribozyme itself can be just z30-40 nt long (Hammann and Lilley 2002). To date, ribozymes constructed via in vitro molecular evolution similar or related to the nucleotide synthetase (Lorsch and Szostak 1994;Unrau and Bartel 1998;Huang 1998;Curtis and Bartel 2005). A shorter nucleotide synthetase ribozyme with a much lower level of efficiency (e.g., 1000-fold as above) does not seem unreasonable.…”

Section: Analysis Of the Resultsmentioning

confidence: 99%

Nucleotide synthetase ribozymes may have emerged first in the RNA world

Chen²,

Zhang³

et al. 2007

RNA

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Though the ''RNA world'' hypothesis has gained a central role in ideas concerning the origin of life, the scenario concerning its emergence remains uncertain. It has been speculated that the first scene may have been the emergence of a template-dependent RNA synthetase ribozyme, which catalyzed its own replication: thus, ''RNA replicase.'' However, the speculation remains uncertain, primarily because of the large sequence length requirement of such a replicase and the lack of a convincing mechanism to ensure its self-favoring features. Instead, we propose a nucleotide synthetase ribozyme as an alternative candidate, especially considering recent experimental evidence suggesting the possibility of effective nonenzymatic templatedirected synthesis of RNA. A computer simulation was conducted to support our proposal. The conditions for the emergence of the nucleotide synthetase ribozyme are discussed, based on dynamic analysis on a computer. We suggest the templatedependent RNA synthetase ribozyme emerged later, perhaps after the emergence of protocells.

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Section: Analysis Of the Resultsmentioning

confidence: 99%

Nucleotide synthetase ribozymes may have emerged first in the RNA world

Chen²,

Zhang³

et al. 2007

RNA

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“…Our questions about adaptive fitness landscapes were addressed in the context of a previously described kinase ribozyme called 5-16 (Curtis and Bartel 2005). This ribozyme thiophosphorylates itself at an internal 2 ′ hydroxyl group using GTPγS as a substrate ( Fig.…”

Section: In Vitro Recombination By Synthetic Shufflingmentioning

confidence: 99%

“…For example, the P4-P6 domain of the Group I intron can fold correctly when separated from the rest of the ribozyme (Murphy and Cech 1993;Cate et al 1996;Doherty and Doudna 1997), and secondary structure elements that can fold and function by themselves are often identified in the context of larger ribozymes by comparative sequence analysis (Ekland and Bartel 1995;Curtis and Bartel 2005). Secondary structure elements can often be broken down into even smaller building blocks.…”

Section: Introductionmentioning

confidence: 99%

“…In this study, we used a method of in vitro recombination called synthetic shuffling (Ness et al 2002), in combination with in vitro selection, comparative sequence analysis, and site-directed mutagenesis, to investigate two related questions about the adaptive fitness landscape of a previously isolated kinase ribozyme (Curtis and Bartel 2005). First, to what extent are mutational effects in this ribozyme independent of one another ( Fig.…”

Section: Introductionmentioning

confidence: 99%

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Synthetic shuffling and in vitro selection reveal the rugged adaptive fitness landscape of a kinase ribozyme

Curtis

Bartel

2013

RNA

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The relationship between genotype and phenotype is often described as an adaptive fitness landscape. In this study, we used a combination of recombination, in vitro selection, and comparative sequence analysis to characterize the fitness landscape of a previously isolated kinase ribozyme. Point mutations present in improved variants of this ribozyme were recombined in vitro in more than 10 14 different arrangements using synthetic shuffling, and active variants were isolated by in vitro selection. Mutual information analysis of 65 recombinant ribozymes isolated in the selection revealed a rugged fitness landscape in which approximately one-third of the 91 pairs of positions analyzed showed evidence of correlation. Pairs of correlated positions overlapped to form densely connected networks, and groups of maximally connected nucleotides occurred significantly more often in these networks than they did in randomized control networks with the same number of links. The activity of the most efficient recombinant ribozyme isolated from the synthetically shuffled pool was 30-fold greater than that of any of the ribozymes used to build it, which indicates that synthetic shuffling can be a rich source of ribozyme variants with improved properties.

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“…Ribozymes and DNAzymes with autophosphorylation activity have been isolated in several laboratories. Some of these molecules catalyze phosphorylation of 59-terminal hydroxyl groups (Lorsch and Szostak 1994;Li and Breaker 1999;Wang et al 2002), while others target internal 29 hydroxyl groups (Lorsch and Szostak 1994;Curtis and Bartel 2005;Saran et al 2005). During the isolation of the Kin.46 ribozyme, the evolving RNA library was incubated with ATPgS.…”

Section: Introductionmentioning

confidence: 99%

Topological rearrangement yields structural stabilization and interhelical distance constraints in the Kin.46 self-phosphorylating ribozyme

Cho¹,

Burke²

2006

RNA

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The Kin.46 ribozyme catalyzes transfer of the gamma (thio)phosphoryl group of ATP (or ATPgS) to the ribozyme's 59 hydroxyl. Single-turnover catalytic activities of topologically rearranged versions of Kin.46 were studied to gain insight into its overall tertiary architecture. The distal ends of stems P3 and P4 were tethered through a single-stranded connection domain that altered the interhelical connectivity. The shortest linkers interfered with catalysis, while seven or more nucleotides (nt) in the linker allowed near-normal catalytic rates, suggesting that a distance of roughly 25-35 Å optimally separates the termini of these helices. Activity was maximal when the tether contained 15 nt, at which point the k cat (0.016 min À1 ) and K m (1.2 mM) values were identical to those of a nontethered control. The presence of the tether alters Mg 2+ dependence, in that Mg 2+ binding appears to be more cooperative in the tethered ribozyme (Hill coefficient 1.4-1.8 versus 0.8 for the nontethered ribozyme). Binding affinity for the ATPgS substrate increases at elevated concentrations of Mg 2+ , particularly for the tethered ribozyme. The tethered ribozyme displays significantly enhanced thermal stability, with a maximum initial velocity (0.126 min À1 ) at 60°C, whereas the nontethered ribozyme has a lower maximum initial velocity (0.051 min À1 ) at 50°C. The tether also significantly reduces the apparent entropy of activation. Both of these effects can be understood in terms of stabilization of the ribozyme in a conformation that is on-path with respect to catalysis, and in terms of facilitating formation of the allosteric activation helix P4.

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New catalytic structures from an existing ribozyme

Cited by 62 publications

References 38 publications

Nucleotide synthetase ribozymes may have emerged first in the RNA world

Nucleotide synthetase ribozymes may have emerged first in the RNA world

Synthetic shuffling and in vitro selection reveal the rugged adaptive fitness landscape of a kinase ribozyme

Topological rearrangement yields structural stabilization and interhelical distance constraints in the Kin.46 self-phosphorylating ribozyme

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