Multiple translational products from a five-nucleotide ribozyme

Abstract

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 (...

Aminoacyl-tRNA synthetase evolution and sectoring of the genetic code

“…Before the code was substantially evolved, it is difficult to imagine tRNA recognition by proteins, and initial aminoacyl transfers may have been catalyzed by ribozymes [20,22,23]. A proposed sequence of events was developed according to the aaRS mechanisms now used to discriminate different archaeal tRNAs.…”

“…At this time, we offer no simple solution to this problem. Ribozymes as small as 5 nt created in vitro can aminoacylate tRNAs [20,22,23], but these ribozyme functions appear now to be fully replaced by aaRS enzymes, so a natural record of aminoacylating ribozymes may not now exist.…”

“…Although the initial evolution of the genetic code may have involved ribozyme-catalyzed tRNA aminoacylation [20–23], at later stages, tRNAs coevolved with aaRS enzymes that attach amino acids at the tRNA 3’-CCA ends [1,2]. Some aaRS enzymes have the capability to proofread tRNA-aa attachments by moving an improperly joined amino acid from the aaRS synthetic site, where the amino acid is linked to the tRNA 3’-CCA end, to a separate aaRS editing or proofreading site, where the non-cognate amino acid is removed [1,2].…”

What can information-asymmetric games tell us about the context of Crick's ‘frozen accident’?

“…Evidence for such a world, for example, in the form of self-aminoacylating RNA [18,19], is growing. However, associated with such a world are a number of complexities, which we have ignored here.…”

Ribozyme Activity of RNA Nonenzymatically Polymerized from 3′,5′-Cyclic GMP

“…This mechanism potentially represents a plausible means to generate RNA sequence complexity and to approach the question: how could the extremely complex ribosomal machinery [31] start to evolve and how did the initial RNA transfer functions [32] come about?…”