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 (...
The RNA world hypothesis requires that early translation be catalyzed by RNA enzymes. Here we show that a five-nucleotide RNA enzyme, reacting with a tetranucleotide substrate and elevated PheAMP, forms aminoacyl- and peptidyl-RNAs RNA-Phe through RNA-Phe(5). A second series of products is formed from RNA-Phe diesters, after trans migration of phenylalanine from the 2'- to the 3'-hydroxyl group of the substrate RNA, followed by reaminoacylation of the 2'-OH. While the ribozyme is required for initial attachment of phenylalanine to an RNA substrate, as well as reacylation (and thus for formation of all products), further extension into RNA-peptides appears to be an uncatalyzed, but RNA-stimulated reaction. The ribozyme readily turns over at high PheAMP and GCCU concentrations. Thus, GUGGC/GCCU comprises a true RNA enzyme. We define Michaelis-Menten parameters plus and minus divalent magnesium and characterize ca. 20 molecular species of aminoacyl-, peptidyl-, dipeptidyl-, and mixed peptidyl/aminoacyl-RNAs.
The invariant choice of L-amino acids and D-ribose RNA for biological translation requires explanation. Here we study this chiral choice using mixed, equimolar D-ribose RNAs having 15, 18, 21, 27, 35, and 45 contiguous randomized nucleotides. These are used for simultaneous affinity selection of the smallest bound and eluted RNAs using equal amounts of L-and D-His immobilized on an achiral glass support, with racemic histidine elution. The experiment as a whole therefore determines whether RNA containing D-ribose binds L-histidine or D-histidine more easily (that is, by using a site that is more abundant/ requires fewer nucleotides). The most prevalent/smallest RNA sites are reproducibly and repeatedly selected and there is a four-to sixfold greater abundance of L-histidine sites. RNA's chiral D-ribose therefore yields a more frequent fit to L-histidine. Accordingly, a D-ribose RNA site for L-His is smaller by the equivalent of just over one conserved nucleotide. The most prevalent L-His site also performs better than the most frequent D-His site-but rarer D-ribose RNAs can bind D-His with excellent affinity and discrimination. The prevalent L-His site is one we have selected before under very different conditions. Thus, selection is again reproducible, as is the recurrence of cognate coding triplets in these most probable L-His sites. If our selected RNA population were equilibrated with racemic His, we calculate that L-His would participate in seven of eight His:RNA complexes, or more. Thus, if D-ribose RNA were first chosen biologically, translational L-His usage could have followed.
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