Specific Interactions of Ribosomes in Decoding

Matthaei, Johannes; Voigt, Hans-Peter; Heller, G; Neth, R.; Schöch, G.; Kübler, Hubert; Amelunxen, F.; Sander, Gernot; Parmeggiani, Andrea

doi:10.1101/sqb.1966.031.01.009

Cited by 32 publications

(13 citation statements)

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“…2). It is known that the stability of the ester link between tRNA and amino acid is determined by the nature of the amino acid attached to the tRNA (Matthaei et al 1966;Drabkin and RajBhandary 1998;Ramesh and RajBhandary 2001), with isoleucine and valine being the most stable, and methionine being much less stable. Aminoacyl-tRNAs present in total RNA were isolated from H. marismortui under acidic conditions to retain the aminoacyl-ester linkage and were subjected to base-catalyzed deacylation (for details, see Materials and Methods).…”

Section: Analysis Ofmentioning

confidence: 99%

Identification and characterization of a tRNA decoding the rare AUA codon in Haloarcula marismortui

Köhrer

Srinivasan²,

Mandal³

et al. 2007

RNA

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Annotation of the complete genome of the extreme halophilic archaeon Haloarcula marismortui does not include a tRNA for translation of AUA, the rare codon for isoleucine. This is a situation typical for most archaeal genomes sequenced to date. Based on computational analysis, it has been proposed recently that a single intron-containing tRNA gene produces two very similar but functionally different tRNAs by means of alternative splicing; a UGG-decoding tRNA Instead, we have shown that a tRNA, currently annotated as elongator methionine tRNA and containing CAU as the anticodon, is aminoacylated with isoleucine in vivo and that this tRNA represents the missing isoleucine tRNA. Interestingly, this tRNA carries a base modification of C34 in the anticodon different from the well-known lysidine found in eubacteria, which switches the amino acid identity of the tRNA from methionine to isoleucine and its decoding specificity from AUG to AUA. The methods described in this work for the identification of individual tRNAs present in H. marismortui provide the tools necessary for experimentally confirming the presence of any tRNA in a cell and, thereby, to test computational predictions of tRNA genes.

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Section: Analysis Ofmentioning

confidence: 99%

Identification and characterization of a tRNA decoding the rare AUA codon in Haloarcula marismortui

Köhrer

Srinivasan²,

Mandal³

et al. 2007

RNA

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“…To investigate whether either of the two CAU anticodon-containing elongator tRNAs, annotated as tRNA_12 and tRNA_34 (The Genomic tRNA Database at http://lowelab.ucsc.edu/GtRNAdb) in H. marismortui, is possibly aminoacylated with isoleucine in vivo, we measured the kinetics of chemical deacylation of aminoacyl-tRNAs corresponding to tRNA_12 and tRNA_34 ( Figure 6A), exploiting the well-known fact that the stability of the ester link between tRNA and amino acid is determined by the nature of the amino acid attached to the tRNA [20]; with isoleucine and valine being the most stable and methionine being much less stable. Aminoacyl-tRNAs present in total RNA were isolated from H. marismortui under acidic conditions to retain the aminoacyl-ester linkage and were subjected to base-catalyzed deacylation.…”

Section: Identification Of the Aua-reading Minor Isoleucine Trna In Amentioning

confidence: 99%

“…• Preparation of bulk deacylated tRNA: A portion of the material is subjected to deacylation by addition of Tris-HCl pH 9.5 to a final concentration of 0.2 M. The deacylation reaction is usually performed at 37 °C for 30−120 min (most tRNAs will be deacylated after 30 min; however, certain tRNAs such as valine and isoleucine tRNAs require longer deacylation treatments [20]); deacylated tRNAs are reprecipitated with ethanol and stored in 10 mM sodium acetate pH 5.0; 10 mM HepesNaOH pH 7.4; or 10 mM Tris pH 7.5 at −80 °C.…”

Section: Deacylation Of Aminoacylated Trna and Measurement Of Deacylamentioning

confidence: 99%

The many applications of acid urea polyacrylamide gel electrophoresis to studies of tRNAs and aminoacyl-tRNA synthetases

Köhrer

RajBhandary

2008

Methods

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Here we describe the many applications of acid urea polyacrylamide gel electrophoresis (acid urea PAGE) followed by Northern blot analysis to studies of tRNAs. Acid urea PAGE allows the electrophoretic separation of different forms of a tRNA, discriminated by changes in bulk, charge, and/or conformation that are brought about by aminoacylation, formylation, or modification of a tRNA. Among the examples described are (i) analysis of the effect of mutations in the Escherichia coli initiator tRNA on its aminoacylation and formylation; (ii) evidence of orthogonality of suppressor tRNAs in mammalian cells and yeast; (iii) analysis of aminoacylation specificity of an archaeal prolyl-tRNA synthetase that can aminoacylate archaeal tRNA Pro with cysteine, but does not aminoacylate archaeal tRNA Cys with cysteine; (iv) identification and characterization of the AUAdecoding minor tRNA Ile in archaea; and (v) evidence that the archaeal minor tRNA Ile contains a modified base in the wobble position different from lysidine found in the corresponding eubacterial tRNA.

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“…This finding was unexpected, suggesting that the determinant for the stability of the acyl linkage was not as simple as the chemical nature of the ester bond. However, some earlier work was performed with unusual components (e.g., 0.5 mM ZnSO 4 ) (Matthaei et al 1966) or at low pH values (e.g., 4.7) (Chousterman et al 1966). In one of the most thorough early analyses, by Hentzen et al (1972), measurements of the acyl linkage stability were performed at pH 7.0 but in buffers with nonphysiological salt concentrations (e.g., 1.5 M potassium phosphate).…”

Section: Introductionmentioning

confidence: 99%

Amino acid–dependent stability of the acyl linkage in aminoacyl-tRNA

Peacock

Walvoord

et al. 2014

RNA

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Aminoacyl-tRNAs are the biologically active substrates for peptide bond formation in protein synthesis. The stability of the acyl linkage in each aminoacyl-tRNA, formed through an ester bond that connects the amino acid carboxyl group with the tRNA terminal 3′ -OH group, is thus important. While the ester linkage is the same for all aminoacyl-tRNAs, the stability of each is not well characterized, thus limiting insight into the fundamental process of peptide bond formation. Here, we show, by analysis of the half-lives of 12 of the 22 natural aminoacyl-tRNAs used in peptide bond formation, that the stability of the acyl linkage is effectively determined only by the chemical nature of the amino acid side chain. Even the chirality of the side chain exhibits little influence. Proline confers the lowest stability to the linkage, while isoleucine and valine confer the highest, whereas the nucleotide sequence in the tRNA provides negligible contribution to the stability. We find that, among the variables tested, the protein translation factor EF-Tu is the only one that can protect a weak acyl linkage from hydrolysis. These results suggest that each amino acid plays an active role in determining its own stability in the acyl linkage to tRNA, but that EF-Tu overrides this individuality and protects the acyl linkage stability for protein synthesis on the ribosome.

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Specific Interactions of Ribosomes in Decoding

Cited by 32 publications

References 0 publications

Identification and characterization of a tRNA decoding the rare AUA codon in Haloarcula marismortui

Identification and characterization of a tRNA decoding the rare AUA codon in Haloarcula marismortui

The many applications of acid urea polyacrylamide gel electrophoresis to studies of tRNAs and aminoacyl-tRNA synthetases

Amino acid–dependent stability of the acyl linkage in aminoacyl-tRNA

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