Enzymatic aminoacylation of single-stranded RNA with an RNA cofactor (original) (raw)
Related papers
Essential structures of a self-aminoacylating RNA 1 1 Edited by D. E. Draper
J Mol Biol, 1997
Comparison of six independent self-aminoacylating RNAs derived from selection-amplification, as well as deletion, addition, substitution, fragmentation of one particular RNA, are used to analyze the requirements for the RNA-catalyzed aminoacylation. All elements required for catalysis by one RNA family: sequence at the 3′ acceptor end, calcium and magnesium sites, as well as the Phe-AMP substrate site and the essential 5′ triphosphate terminus, are closely grouped near a bihelix junction in the parental molecule. All elements of the active center for aminoacyl transfer can therefore be captured by a peripherally-deleted helix junction RNA, defining a much smaller 43 nucleotide ribozyme, of which only 17 nucleotides were initially randomized. It appears that a complex RNA active center can be assembled by specifying unexpectedly few nucleotides, perhaps with a critical contribution from an essential calcium ion.
Essential structures of a self-aminoacylating RNA
Journal of Molecular Biology, 1997
Comparison of six independent self-aminoacylating RNAs derived from selection-ampli®cation, as well as deletion, addition, substitution, fragmentation of one particular RNA, are used to analyze the requirements for the RNA-catalyzed aminoacylation. All elements required for catalysis by one RNA family: sequence at the 3 H acceptor end, calcium and magnesium sites, as well as the Phe-AMP substrate site and the essential 5 H triphosphate terminus, are closely grouped near a bihelix junction in the parental molecule. All elements of the active center for aminoacyl transfer can therefore be captured by a peripherally-deleted helix junction RNA, de®ning a much smaller 43 nucleotide ribozyme, of which only 17 nucleotides were initially randomized. It appears that a complex RNA active center can be assembled by specifying unexpectedly few nucleotides, perhaps with a critical contribution from an essential calcium ion.
European Journal of Biochemistry, 1992
RNA microhelices that reconstruct the acceptor stems of transfer RNAs can be aminoacylated. The anticodon-independent aminoacylation is sequence-specific and suggests a relationship between amino acids and nucleotide sequences which is different from that of the classical genetic code. The specific aminoacylation of RNA microhelices also suggests a highly differentiated adaptation of the structures of aminoacyl-tRNA synthetases to sequences in the acceptor stems of transfer RNAs.
The EMBO journal, 1994
We show here that small RNA helices which recapitulate part or all of the acceptor stem of yeast aspartate tRNA are efficiently aminoacylated by cognate class II aspartyl-tRNA synthetase. Aminoacylation is strongly dependent on the presence of the single-stranded G73 'discriminator' identity nucleotide and is essentially insensitive to the sequence of the helical region. Substrates which contain as few as 3 bp fused to G73CCAOH are aspartylated. Their charging is insensitive to the sequence of the loop closing the short helical domains. Aminoacylation of the aspartate mini-helix is not stimulated by a hairpin helix mimicking the anticodon domain and containing the three major anticodon identity nucleotides. A thermodynamic analysis demonstrates that enzyme interactions with G73 in the resected RNA substrates and in the whole tRNA are the same. Thus, if the resected RNA molecules resemble in some way the earliest substrates for aminoacylation with aspartate, then the contempo...
Ribozyme-catalyzed tRNA aminoacylation
Nature structural biology, 2000
The RNA world hypothesis implies that coded protein synthesis evolved from a set of ribozyme catalyzed acyl-transfer reactions, including those of aminoacyl-tRNA synthetase ribozymes. We report here that a bifunctional ribozyme generated by directed in vitro evolution can specifically recognize an activated glutaminyl ester and aminoacylate a targeted tRNA, via a covalent aminoacyl-ribozyme intermediate. The ribozyme consists of two distinct catalytic domains; one domain recognizes the glutamine substrate and self-aminoacylates its own 5'-hydroxyl group, and the other recognizes the tRNA and transfers the aminoacyl group to the 3'-end. The interaction of these domains results in a unique pseudoknotted structure, and the ribozyme requires a change in conformation to perform the sequential aminoacylation reactions. Our result supports the idea that aminoacyl-tRNA synthetase ribozymes could have played a key role in the evolution of the genetic code and RNA-directed translation.
Small-molecule-substrate interactions with a self-aminoacylating ribozyme
Journal of Molecular Biology, 1997
A self-aminoacylating RNA catalyst is shown to carry out the chemistry required for turnover, being reacylated several times from aminoacyl-AMP with an unaltered rate, thereby meeting one de®nition of an enzyme. Furthermore, a newly applied gel electrophoresis assay suggests ®rst order kinetics in RNA and saturation kinetics in the substrate aminoacyl-adenylate, implying a Michaelis complex. AMP is a competitive inhibitor, though phenylalanine is not detectably inhibitory, consistent with a Michaelis complex through the AMP moiety of phenylalanyladenylate substrate. This idea is supported by measurement of elevated acylation velocities with seryl and alanyl-adenylates. The rate of aminoacylation increases with pH, consistent with attack of a terminal ribose oxyanion on the carbonyl carbon atom of the adenylate.
RNA-Catalyzed Amino Acid Activation †
Biochemistry, 2001
We have selected RNAs that perform a new reaction that chemically activates amino acids, paralleling mixed phosphate anhydride synthesis by protein aminoacyl-transfer RNA synthetases. Care with recovery of the unstable reaction product was apparently essential to this selection. The best characterized RNA, KK13, requires only Ca 2+ for reaction and is optimally active at low pH with K M ) 50 mM and k cat ) 1.1 min -1 for activation of leucine. In conjunction with previous RNA-catalyzed aminoacyl-RNA synthesis, peptide bond formation, and RNA-based coding, these amino acid-activating RNAs complete an experimental demonstration that the four fundamental reactions of protein biosynthesis can be RNA-mediated. The appearance of translation in an RNA world is therefore supported.
Multiple translational products from a five-nucleotide ribozyme
Proceedings of the National Academy of Sciences, 2010
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.