Rapid and Simple Ribozymic Aminoacylation Using Three Conserved Nucleotides

Chumachenko, Nataliya; Novikov, Yehor; Yarus, Michael

doi:10.1021/ja809419f

Cited by 64 publications

(90 citation statements)

References 34 publications

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“…In addition, chemically capable RNAs can be very small. A pentaribonucleotide ribozyme suffices to perform highly regiospecific aminoacyl transfer (Chumachenko et al 2009;Yarus 2011b). RNAs as small as two nucleotides have chemical phenotypes, even today (Yarus 2011a).…”

Section: Introductionmentioning

confidence: 99%

Cross-backbone templating; ribodinucleotides made on poly(C)

Majerfeld

Puthenvedu

Yarus

2016

RNA

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′ G synthesis from pG and chemically activated 2MeImpG is accelerated by the addition of complementary poly(C), but affected only slightly by poly(G) and not at all by poly(U) and poly(A). This suggests that 3 ′ -5 ′ poly(C) is a template for uncatalyzed synthesis of 5 ′ -5 ′ GppG, as was poly(U) for AppA synthesis, previously. The reaction occurs at 50 mM monoand divalent ion concentrations, at moderate temperatures, and near pH 7. The reactive complex at the site of enhanced synthesis of 5 ′ -5 ′ GppG seems to contain a single pG, a single phosphate-activated nucleotide 2MeImpG, and a single strand of poly(C). Most likely this structure is base-paired, as the poly(C)-enhanced reaction is completely disrupted between 30 and 37°C, whereas slower, untemplated synthesis of GppG accelerates. More specifically, the reactive center acts as would be expected for short, isolated G nucleotide stacks expanded and ordered by added poly(C). For example, poly(C)-mediated GppG production is very nonlinear in overall nucleotide concentration. Uncatalyzed NppN synthesis is now known for two polymers and their complementary free nucleotides. These data suggest that varied, simple, primordial 3 ′ -5 ′ RNA sequences could express a specific chemical phenotype by encoding synthesis of complementary, reactive, coenzyme-like 5 ′ -5 ′ ribodinucleotides.

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Section: Introductionmentioning

confidence: 99%

Cross-backbone templating; ribodinucleotides made on poly(C)

Majerfeld

Puthenvedu

Yarus

2016

RNA

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“…In addition, it should act in trans, and should use universal biological, water-soluble substrates. Previously isolated ribozymes employ appropriate substrates [amino acids activated as acyladenylates (6,12)], but are not true enzymes, as they are self-aminoacylators modified by their own reaction. Other ribozymes aminoacylate RNA with turnover; however, the amino acids must be activated as cyanomethyl, 3,5-dinitrobenzyl, or p-chlorobenzyl esters (8,13), and hence they do not facilitate the biological reaction.…”

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confidence: 99%

“…1A) presented the intriguing possibility of very simple aminoacyl transfer (6). Mutational analyses as well as molecular dynamics and energy minimization of the reactants suggested a tiny active center consisting of only three essential nucleotides-a 3 0 -terminal U, and a 5 0 -GU-3 0 sequence in a loop apposed to the unpaired 3 0 -terminal U.…”

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confidence: 99%

Multiple translational products from a five-nucleotide ribozyme

Turk

Chumachenko

Yarus

2010

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

146

145

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An indispensable step in protein biosynthesis is the 2 0 ð3 0 Þ aminoacylation of tRNA by aminoacyl-tRNA synthetases. Here we show that a similar activity exists in a tiny, 5-nt-long RNA enzyme with a 3-nt active center. The small ribozyme initially trans-phenylalanylates a partially complementary 4-nt RNA selectively at its terminal 2 0 -ribose hydroxyl using PheAMP, the natural form for activated amino acid. The initial 2 0 Phe-RNA product can be elaborated into multiple peptidyl-RNAs. Reactions do not require divalent cations, and have limited dependence on monovalent cations. Small size and minimal requirements for regiospecific translational activity strongly support the hypothesis that minuscule RNA enzymes participated in early forms of translation.aminoacyl-RNA | enzyme | evolution | peptidyl-RNA | RNA A mino acids enter modern translation via attachment to a 2 0 ð3 0 Þ tRNA terminus, a reaction catalyzed by a protein aminoacyl-tRNA synthetase. Because it is implausible that primitive peptides were synthesized using already-formed protein catalysts, the RNA world hypothesis (1, 2) requires peptide synthetic reactions performed by RNA enzymes (3, 4). Indeed, a number of RNAs have been isolated which accelerate related translational reactions (5).Several ribozymes capable of catalyzing the same chemical group transfer that is today carried out by aminoacyl-tRNA synthetases (6-9) have been isolated using Systematic Evolution of Ligands by Exponential Enrichment (10, 11). However, none possess all desirable characteristics. First, an RNA world enzyme should be small, accessible after rudimentary RNA synthesis. In addition, it should act in trans, and should use universal biological, water-soluble substrates. Previously isolated ribozymes employ appropriate substrates [amino acids activated as acyladenylates (6, 12)], but are not true enzymes, as they are self-aminoacylators modified by their own reaction. Other ribozymes aminoacylate RNA with turnover; however, the amino acids must be activated as cyanomethyl, 3,5-dinitrobenzyl, or p-chlorobenzyl esters (8, 13), and hence they do not facilitate the biological reaction.The family of self-aminoacylating ribozymes exemplified by truncate C3 RNA (Fig. 1A) presented the intriguing possibility of very simple aminoacyl transfer (6). Mutational analyses as well as molecular dynamics and energy minimization of the reactants suggested a tiny active center consisting of only three essential nucleotides-a 3 0 -terminal U, and a 5 0 -GU-3 0 sequence in a loop apposed to the unpaired 3 0 -terminal U. Although the C3 RNA family possessed helical elements, adjacent helices appeared nonspecific in sequence, perhaps required only for assembly of the active center (6).Here we present a radically modified version of C3 ribozyme, unique in three ways: It functions in trans; it has been minimized to a tiny, five-nucleotide ribozyme; and it also supports peptidyl-RNA synthesis. The result is the smallest trans-aminoacylator, and arguably the smallest true ribozyme, ever observed (...

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“…16 Other ribozymes that catalyze cis-aminoacylation or trans-aminoacylation on the 3¤-end of RNA in the presence of Phe-AMP (Phe: phenylalanine) were also generated. 12,17 Although the above ribozymes partly mimic the function of ARSs, they were unable to charge onto tRNA. Bessho et al have reported an acyl-transferase ribozyme Figure 1.…”

Section: Rna-acylation Ribozymes Flexizymesmentioning

confidence: 99%

Flexizymes-facilitated Genetic Code Reprogramming Leading to the Discovery of Drug-like Peptides

Terasaka

Suga

2013

Chemistry Letters

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Flexizymes are a family of aminoacylation ribozymes devised by in vitro selection in our research group. They charge a wide variety of nonproteinogenic amino acids onto virtually any kind of tRNAs, enabling us to reprogram the genetic code in a custom-made cell-free translation system. This genetic code reprogramming method was integrated with a mRNA display method, which not only expresses nonstandard peptides such as macrocyclic peptides but also selects ligands specifically binding to target proteins. This system is referred to as random nonstandard peptide integrated discovery (RaPID) system, which has yielded inhibitors against disease-related target proteins. In this review, we summarize the evolutionary history and recent applications of the flexizymes and RaPID system.

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Rapid and Simple Ribozymic Aminoacylation Using Three Conserved Nucleotides

Cited by 64 publications

References 34 publications

Cross-backbone templating; ribodinucleotides made on poly(C)

Cross-backbone templating; ribodinucleotides made on poly(C)

Multiple translational products from a five-nucleotide ribozyme

Flexizymes-facilitated Genetic Code Reprogramming Leading to the Discovery of Drug-like Peptides

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