Oligomerization of (guanosine 5′-phosphor)-2-methylimidazolide on poly(C)

Inoue, Tan; Orgel, Leslie E.

doi:10.1016/0022-2836(82)90169-3

Cited by 198 publications

(137 citation statements)

References 15 publications

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“…Though higher than the 0.25 fidelity of polymerization expected from random extension, the observed fidelity of primer extension catalyzed by the Tetrahymena ribozyme is far lower than that required for an RNA replicase. Our results are consistent with the earlier results of Orgel and colleagues (4,(13)(14)(15) …”

supporting

confidence: 94%

Template-directed primer extension catalyzed by the Tetrahymena ribozyme.

et al. 1991

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The Tetrahymena ribozyme has been shown to catalyze an RNA polymerase-like reaction in which an RNA primer is extended by the sequential addition of pN nucleotides derived from GpN dinucleotides, where N = A, C, or U. Here, we show that this reaction is influenced by the presence of a template; bases that can form Watson-Crick base pairs with a template add as much as 25-fold more efficiently than mismatched bases. A mutant enzyme with an altered guanosine binding site can catalyze template-directed primer extension with all four bases when supplied with dinucleotides of the form 2-aminopurine-pN.Our interest in the possible role of RNA replicases in the origin and early evolution of life (2,5,8,12,16,24) led us to examine the extent to which a template can direct the incorporation of mononucleotides in a primer extension reaction catalyzed by the Tetrahymena ribozyme (1). The primer extension reaction involves nucleophilic attack by the 3' hydroxyl group of an oligonucleotide primer on the phosphate of a GpN (where N = A, C, or U) dinucleotide. This reaction extends the primer by one nucleotide and releases the guanosine nucleoside ( Fig. la). Each addition is analogous to the second step of self-splicing (6), in which the primer represents the 5' exon and is extended by a singlenucleotide 3' exon (17). This primer extension reaction is also analogous to protein-catalyzed RNA and DNA polymerization in that the 3' end of the growing chain is extended by successive additions of mononucleotides, with guanosine as the leaving group instead of pyrophosphate.Template-influenced primer extension. The original primerextension system studied by Been and Cech (1) did not include a uniquely defined template base that could interact with the incoming nucleotide. We tested the possibility that such a template base might influence the primer extension reaction by adding a template region just 5' of the primerbinding site, as illustrated in Fig. la. In our scheme, the primer can anneal with the primer-binding site in only one way; since the primer cannot slide along the template, it is possible to examine the influence of a single template base.Four ribozymes that differed in the template base were incubated with dinucleotide and labeled primer, with enzyme in large excess over primer (Fig. lb). All four ribozymes begin with pppG, followed by the indicated template base, followed by the primer-binding site, followed by the catalytic core (bases G27 to G405 in reference 7) of the wild-type Tetrahymena intron. These ribozymes were generated by runoff transcription of plasmid templates that were linearized at an EcoRI site inserted immediately after the position corresponding to G405 of the wild-type intron. Plasmids were constructed by polymerase chain reaction mutagenesis (21), and transcription and purification of ribozymes were as previously described (19). Primer was transcribed from a single-stranded oligonucleotide template * Corresponding author.(20). Dinucleotide initiation (22) with 4 mM GpC and abortive cycling (18) w...

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supporting

confidence: 94%

Template-directed primer extension catalyzed by the Tetrahymena ribozyme.

et al. 1991

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“…The 2′-5′ linkage is an alternative form of nucleic acid backbone in addition to the 3′-5′ linkage during molecular evolution (42,43). Previous studies have demonstrated that a mixture of 2′-5′ and 3′-5′ phosphodiester linkage can be obtained during nonenzymatic RNA replication, suggesting a high probability of mixture of phosphodiester linkages in the early evolution of life (3)(4)(5)(6)(7)(8)(9). It is conceivable that RNA in early evolution of life may have contained a mixture of 2′-5′ and 3′-5′ phosphodiester linkages.…”

Section: Insights Into Molecular Evolution Of Phosphodiester Linkagementioning

confidence: 99%

“…Indeed, previous studies revealed that nonenzymatic RNA replication would lead to a mixture of 2′-5′ and 3′-5′ linkages (Fig. 1A) (3)(4)(5)(6)(7)(8)(9). The 2′-5′-linked nucleic acids can form duplex structures by themselves or by pairing with natural nucleic acids (10)(11)(12)(13)(14)(15).…”

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confidence: 96%

Strand-specific (asymmetric) contribution of phosphodiester linkages on RNA polymerase II transcriptional efficiency and fidelity

Zhang

Chong

et al. 2014

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

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Nonenzymatic RNA polymerization in early life is likely to introduce backbone heterogeneity with a mixture of 2′-5′ and 3′-5′ linkages. On the other hand, modern nucleic acids are dominantly composed of 3′-5′ linkages. RNA polymerase II (pol II) is a key modern enzyme responsible for synthesizing 3′-5′-linked RNA with high fidelity. It is not clear how modern enzymes, such as pol II, selectively recognize 3′-5′ linkages over 2′-5′ linkages of nucleic acids. In this work, we systematically investigated how phosphodiester linkages of nucleic acids govern pol II transcriptional efficiency and fidelity. Through dissecting the impacts of 2′-5′ linkage mutants in the pol II catalytic site, we revealed that the presence of 2′-5′ linkage in RNA primer only modestly reduces pol II transcriptional efficiency without affecting pol II transcriptional fidelity. In sharp contrast, the presence of 2′-5′ linkage in DNA template leads to dramatic decreases in both transcriptional efficiency and fidelity. These distinct effects reveal that pol II has an asymmetric (strand-specific) recognition of phosphodiester linkage. Our results provided important insights into pol II transcriptional fidelity, suggesting essential contributions of phosphodiester linkage to pol II transcription. Finally, our results also provided important understanding on the molecular basis of nucleic acid recognition and genetic information transfer during molecular evolution. We suggest that the asymmetric recognition of phosphodiester linkage by modern nucleic acid enzymes likely stems from the distinct evolutionary pressures of template and primer strand in genetic information transfer during molecular evolution.2′-5′ phosphodiester linkage | trigger loop | synthetic nucleic acid analogues T he remarkable capacity of RNA as the functional catalyst as well as the genetic information carrier provides a strong support for the RNA world hypothesis, in which RNA is proposed to play a key role in the early evolution of primitive life before DNA and protein (enzyme) evolved (1, 2). One critical issue for RNA replication in early life is backbone heterogeneity because RNA contains both 2′-OH and 3′-OH, in which both can form phosphodiester bond during RNA polymerization. Indeed, previous studies revealed that nonenzymatic RNA replication would lead to a mixture of 2′-5′ and 3′-5′ linkages (Fig. 1A) (3-9). The 2′-5′-linked nucleic acids can form duplex structures by themselves or by pairing with natural nucleic acids (10-15). The 2′-5′ linkage substitutions in some functional RNAs retain molecular recognition and catalytic properties (16)(17)(18)(19). These discoveries suggested that the 2′-5′ linkage in nucleic acids could be an important alternative linkage during early evolution.This backbone heterogeneity problem is largely eliminated during evolution. Modern nucleic acids are dominated by 3′-5′ linkages. The 2′-5′ RNA linkages are only present in a few specific processes. One example is the formation of lariat RNA intron during splicing, where the 2′-OH of an...

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“…In the absence of complex macromolecular catalysts, new internucleotide linkages are inevitably forged as both 3′,5′‐ and 2′,5′‐phosphodiester bonds 6, 7, 8. The problem has prompted extensive investigation, ranging from synthetic efforts to favor natural 3′,5′‐bonds,8, 9, 10, 11 to the finding that, below a threshold level, backbone heterogeneity might be compatible with the catalytic and recognition properties of RNA 12. Nevertheless, the question of how RNA might have evolved to the exclusively 3′,5′‐linked material that is primarily employed by extant biology remains largely unanswered.…”

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confidence: 99%

Non‐Enzymatic RNA Backbone Proofreading through Energy‐Dissipative Recycling

Mariani

Sutherland

2017

Angew Chem Int Ed

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Non‐enzymatic oligomerization of activated ribonucleotides leads to ribonucleic acids that contain a mixture of 2′,5′‐ and 3′,5′‐linkages, and overcoming this backbone heterogeneity has long been considered a major limitation to the prebiotic emergence of RNA. Herein, we demonstrate non‐enzymatic chemistry that progressively converts 2′,5′‐linkages into 3′,5′‐linkages through iterative degradation and repair. The energetic costs of this proofreading are met by the hydrolytic turnover of a phosphate activating agent and an acylating agent. With multiple rounds of this energy‐dissipative recycling, we show that all‐3′,5′‐linked duplex RNA can emerge from a backbone heterogeneous mixture, thereby delineating a route that could have driven RNA evolution on the early earth.

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Oligomerization of (guanosine 5′-phosphor)-2-methylimidazolide on poly(C)

Cited by 198 publications

References 15 publications

Template-directed primer extension catalyzed by the Tetrahymena ribozyme.

Template-directed primer extension catalyzed by the Tetrahymena ribozyme.

Strand-specific (asymmetric) contribution of phosphodiester linkages on RNA polymerase II transcriptional efficiency and fidelity

Non‐Enzymatic RNA Backbone Proofreading through Energy‐Dissipative Recycling

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