Characterization of the B6.61 polymerase ribozyme accessory domain

Wang, Qing S.; Cheng, L.; Unrau, Peter J.

doi:10.1261/rna.2495011

Cited by 20 publications

(32 citation statements)

References 22 publications

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“…An engineered form of the class I polymerase ribozyme [wild type (WT); Fig. S1] was constructed by combining known advantageous features, including a large deletion within the 3′-terminal domain; a 5′-terminal sequence tag that binds to a complementary region of the template; and a set of activity-enhancing mutations (14,16). Random mutations then were introduced throughout the molecule at a frequency of 10% per nucleotide position to generate a population of 10 14 distinct variants to initiate the in vitro evolution process.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Amplification of RNA by an RNA polymerase ribozyme

Horning

Joyce

2016

Proc. Natl. Acad. Sci. U.S.A.

222

244

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In all extant life, genetic information is stored in nucleic acids that are replicated by polymerase proteins. In the hypothesized RNA world, before the evolution of genetically encoded proteins, ancestral organisms contained RNA genes that were replicated by an RNA polymerase ribozyme. In an effort toward reconstructing RNA-based life in the laboratory, in vitro evolution was used to improve dramatically the activity and generality of an RNA polymerase ribozyme by selecting variants that can synthesize functional RNA molecules from an RNA template. The improved polymerase ribozyme is able to synthesize a variety of complex structured RNAs, including aptamers, ribozymes, and, in low yield, even tRNA. Furthermore, the polymerase can replicate nucleic acids, amplifying short RNA templates by more than 10,000-fold in an RNA-catalyzed form of the PCR. Thus, the two prerequisites of Darwinian life-the replication of genetic information and its conversion into functional molecules-can now be accomplished with RNA in the complete absence of proteins.self-replication | ribozyme | RNA enzyme | PCR | in vitro evolution T he core informational processes of Darwinian evolution are the replication of genes and their expression as functional molecules, which in modern biology require the action of protein enzymes. Features common to all extant life suggest the existence of an RNA world where the replication and expression of genetic information depended on RNA enzymes rather than genetically encoded proteins (1, 2). Accordingly, substantial efforts have been directed toward reconstructing RNA life via the protein-free replication of RNA (3, 4). RNA-joining ribozymes have been modified to assemble new copies of themselves from smaller RNA substrates (5), in one case achieving self-replication with exponential growth (6). However, these self-replicating ribozymes require complex oligonucleotide substrates, which limits their ability to synthesize functional RNA molecules other than additional copies of themselves, which in turn limits their ability to express and evolve functions beyond self-replication.A more general solution is offered by the template-directed polymerization of RNA monomers, the mechanism used in modern biology for the synthesis of DNA and RNA. RNAtemplated RNA polymerization has been demonstrated both nonenzymatically (7,8) and with a variety of natural and synthetic ribozymes (9-11). Although none of these systems have been able to replicate RNA exponentially, extensive copying of RNA templates has been achieved with evolved variants of the class I RNA polymerase ribozyme. This ribozyme can synthesize tandem RNA repeats over 100 nt long and even complete a substantial portion of a small endonuclease ribozyme (12-15). However, the polymerase strongly prefers cytidine-rich templates that lack any secondary structure and has much more limited activity in other contexts (15). These limitations preclude the synthesis of most functional RNAs, which often are highly structured, and the replication of RNA, which re...

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Section: Resultsmentioning

confidence: 99%

“…All examples to date, however, have suffered from slow rates and a strong preference for unstructured, C-rich templates (12)(13)(14)(15)(16). In vitro evolution was used to select polymerase variants that can synthesize functional RNA aptamers, resulting in dramatically improved activity and sequence generality.…”

Section: Discussionmentioning

confidence: 99%

Amplification of RNA by an RNA polymerase ribozyme

Horning

Joyce

2016

Proc. Natl. Acad. Sci. U.S.A.

222

244

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“…Instead, the pyrophosphate leaving group is oriented back into the major groove, and nucleobase and hydroxyl groups are thought to stabilize the transition state electrostatically through hydrogen bonding. The accessory domain is less well characterized, but Wang et al (105) (107) evolved the hc ligase into the 18-2 ribozyme, which can catalyze the addition of single nucleotides to a primer by using NTPs as substrates. Other natural (108) and selected (109) ligase ribozymes could be evolved into polymerases; however, the direct selection for polymerase ribozymes with high affinity for the primer-template substrate would avoid the difficulty of evolving an RNA that was initially selected to perform one function into a variant with a different function.…”

Section: Ribozyme-catalyzed Replicationmentioning

confidence: 99%

Progress Toward Synthetic Cells

Blain

Szostak²

2014

Annu. Rev. Biochem.

275

249

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The complexity of even the simplest known life forms makes efforts to synthesize living cells from inanimate components seem like a daunting task. However, recent progress toward the creation of synthetic cells, ranging from simple protocells to artificial cells approaching the complexity of bacteria, suggests that the synthesis of life is now a realistic goal. Protocell research, fueled by advances in the biophysics of primitive membranes and the chemistry of nucleic acid replication, is providing new insights into the origin of cellular life. Parallel efforts to construct more complex artificial cells, incorporating translational machinery and protein enzymes, are providing information about the requirements for protein-based life. We discuss recent advances and remaining challenges in the synthesis of artificial cells, the possibility of creating new forms of life distinct from existing biology, and the promise of this research for gaining a deeper understanding of the nature of living systems.

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“…Secondary structure of the polymerase ribozyme Pol1e. The secondary structure is based on Johnston et al (2001), Shechner et al (2009), andWang et al (2011). (Blue) The wild-type P2 oligo; (green) the primer/template substrate with the sequence of template T21 and the corresponding primer.…”

Section: Truncations Of P2 Oligonucleotidesmentioning

confidence: 99%

“…(Arrowheads) Residues close to the catalytic site. These are the unpaired C in helix P4 (Shechner et al 2009), the 39-terminus of the P2 oligo (Ekland and Bartel 1996), and the loop connecting helices A3 and A4 (Wang et al 2011). (Dotted lines) The tertiary contacts between the accessory domain and ligase core (Wang et al 2011).…”

Section: Truncations Of P2 Oligonucleotidesmentioning

confidence: 99%

Polymerase ribozyme efficiency increased by G/T-rich DNA oligonucleotides

Yao

Müller²

2011

RNA

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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 39-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.

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Characterization of the B6.61 polymerase ribozyme accessory domain

Cited by 20 publications

References 22 publications

Amplification of RNA by an RNA polymerase ribozyme

Amplification of RNA by an RNA polymerase ribozyme

Progress Toward Synthetic Cells

Polymerase ribozyme efficiency increased by G/T-rich DNA oligonucleotides

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