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Multiple translational products from a five-nucleotide ribozyme - PubMed

  • ️Fri Jan 01 2010

Multiple translational products from a five-nucleotide ribozyme

Rebecca M Turk et al. Proc Natl Acad Sci U S A. 2010.

Abstract

An indispensable step in protein biosynthesis is the 2(')(3(')) 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(')-ribose hydroxyl using PheAMP, the natural form for activated amino acid. The initial 2(') 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.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.

Small trans-aminoacylating RNA complexes. (A) C3 RNA. (B) Intermediate trans complexes. (C) Final GUGGC/GCCU complex.

Fig. 2.
Fig. 2.

Initial reaction occurs only at 2′-OH. Lanes: 1, substrate = GCCU; 2, GCC2′dU; 3, GCC3′dU; 4, GCCU + NaIO4; 5, GCC; 6, dGdCdCrU; 7, GCCU + PheAMP, no GUGGC; 8, GCCU + GUGGC, no PheAMP. Reaction conditions: 10 μM ribozyme GUGGC, 20 μM RNA substrate, 100 mM KCl, 5 mM MgCl2, 100 mM Hepes pH 7.0, 2.0 mM PheAMP; incubation at 4 °C for 30 min.

Fig. 3.
Fig. 3.

Aminoacylation reaction does not require divalent cations. All reactions contained 10 μM GUGGC, 20 μM GCCU, 1.9 mM PheAMP, 100 mM Hepes pH 6.5, 10 mM NaOH. Reactions contained 100 mM KCl, 5 mM MgCl2, or 100 mM NaCl where indicated. Incubation at 4 °C for 60 min.

Fig. 4.
Fig. 4.

MALDI-TOF mass spectrometric analysis of RNA and Products 1 and 2. (A) Product 1 and RNA. (B) Product 2. Numbers in parentheses indicate theoretical mass of protonated products; outside parentheses, observed mass.

Fig. 5.
Fig. 5.

Higher products are RNA peptides. Aminoacylation reaction conditions: 10 μM GUGGC, 20 μM GCCU, 100 mM KCl, 5 mM MgCl2, 100 mM Hepes pH 7.0, 1 mM PheAMP; incubation at 4 °C for 30 min. LAP, leucine aminopeptidase, 23 ng/μL.

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