Preparation of misacylated aminoacyl-tRNAPhe's useful as probes of the ribosomal acceptor site

Roesser, James R.; Xu, Cheng; Payne, Robert C.; Surratt, Christopher K.; Hecht, Sidney M.

doi:10.1021/bi00438a041

Cited by 68 publications

(43 citation statements)

References 61 publications

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“…Our preliminary results, however, suggest that most, if not all, of the side-chain analogs that we studied are translated with high efficiency. Based on several reports, we expect that the ␤-amino acids will not be translated (56)(57)(58). Conversely, at least some of the N-Me amino acid AARS substrates should be efficient translation substrates (8,(58)(59)(60).…”

Section: Discussionmentioning

confidence: 99%

Enzymatic aminoacylation of tRNA with unnatural amino acids

Hartman

Josephson²,

Szostak³

2006

Proc. Natl. Acad. Sci. U.S.A.

139

172

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The biochemical flexibility of the cellular translation apparatus offers, in principle, a simple route to the synthesis of drug-like modified peptides and novel biopolymers. However, only Ϸ75 unnatural building blocks are known to be fully compatible with enzymatic tRNA acylation and subsequent ribosomal synthesis of modified peptides. Although the translation system can reject substrate analogs at several steps along the pathway to peptide synthesis, much of the specificity resides at the level of the aminoacyl-tRNA synthetase (AARS) enzymes that are responsible for charging tRNAs with amino acids. We have developed an AARS assay based on mass spectrometry that can be used to rapidly identify unnatural monomers that can be enzymatically charged onto tRNA. By using this assay, we have found 59 previously unknown AARS substrates. These include numerous side-chain analogs with useful functional properties. Remarkably, many ␤-amino acids, N-methyl amino acids, and ␣,␣-disubstituted amino acids are also AARS substrates. These previously unidentified AARS substrates will be useful in studies of the specificity of subsequent steps in translation and may significantly expand the number of analogs that can be used for the ribosomal synthesis of modified peptides.aminoacyl-tRNA synthetases ͉ enzyme specificity ͉ MALDI-TOF mass spectrometry ͉ translation

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Section: Discussionmentioning

confidence: 99%

Enzymatic aminoacylation of tRNA with unnatural amino acids

Hartman

Josephson²,

Szostak³

2006

Proc. Natl. Acad. Sci. U.S.A.

139

172

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“…This mischarging event gives a negative impact on cell growth via two possible mechanisms. A small portion of such a mischarged D aa may be incorporated into a nascent peptide chain even though D aa is generally a poor substrate for elongation (Roesser et al 1989;Bain et al 1991;Ellman et al 1992;Dedkova et al 2003;Murakami et al 2006). Alternatively, undesirable generation of the mischarged elongator tRNAs may decrease the concentration of available elongator tRNAs for the innate function.…”

Section: Discussionmentioning

confidence: 99%

“…However, even if the tRNA aminoacylation step is circumvented, D aa cannot be efficiently incorporated into the nascent peptide chain during elongation. For instance, a variety of D aa precharged onto an ''amber'' suppressor tRNA CUA have been examined for elongation; they are either modestly or in many cases not at all incorporated into the nascent peptide chain (Roesser et al 1989;Bain et al 1991;Ellman et al 1992;Starck et al 2003;Tan et al 2004;Murakami et al 2006). This has been attributed to the fact that either elongation factor (EF-Tu) or ribosome (or possibly both) does not allow D aa-tRNA CUA to read the amber stop codon, resulting in the undesired termination of peptide synthesis executed by release factor.…”

Section: Introductionmentioning

confidence: 99%

Initiating translation with D-amino acids

Murakami¹,

Suga²

2008

RNA

115

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Here we report experimental evidence that the translation initiation apparatus accepts D-amino acids ( D aa), as opposed to only L-methionine, as initiators. Nineteen D aa, as the stereoisomers to their natural L-amino acids, were charged onto initiator tRNA fMet CAU using flexizyme technology and tested for initiation in a reconstituted Escherichia coli translation system lacking methionine, i.e., the initiator was reprogrammed from methionine to D aa. Remarkably, all D aa could initiate translation while the efficiency of initiation depends upon the type of side chain. The peptide product initiated with D aa was generally in a nonformylated form, indicating that methionyl-tRNA formyltransferase poorly formylated the corresponding

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“…After phenol-chloroform extraction and ethanol precipitation, the resulting total RNA was dissolved in 40 μL of H 2 O. One quarter of the extracted RNA was incubated in 50 μL of 150 mM NaOAc pH 5.5 containing 10 mM CuSO 4 as described (Roessler et al 1989), ethanol-precipitated, dissolved in 20 μL of H 2 O and 3 ′ end-labeled with 20 μCi of [5 ′ -32 P] pCp using T4 RNA ligase. The rest of the total RNA obtained from the 80S-II complex was incubated in 50 mM Tris-HCl pH 9.0 for 1 h at 25°C, precipitated, and subjected to 3 ′ end labeling, too.…”

Section: Trnamentioning

confidence: 99%

A novel insight into the mechanism of mammalian selenoprotein synthesis

Kossinova

Malygin

Krol

et al. 2013

RNA

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The amino acid selenocysteine is encoded by UGA, usually a stop codon, thus requiring a specialized machinery to enable its incorporation into selenoproteins. The machinery comprises the tRNA Sec , a 3 ′ -UTR mRNA stem-loop termed SElenoCysteine Insertion Sequence (SECIS), which is mandatory for recoding UGA as a Sec codon, the SECIS Binding Protein 2 (SBP2), and other proteins. Little is known about the molecular mechanism and, in particular, when, where, and how the SECIS and SBP2 contact the ribosome. Previous work by others used the isolated SECIS RNA to address this question. Here, we developed a novel approach using instead engineered minimal selenoprotein mRNAs containing SECIS elements derivatized with photoreactive groups. By cross-linking experiments in rabbit reticulocyte lysate, new information could be gained about the SBP2 and SECIS contacts with components of the translation machinery at various translation steps. In particular, we found that SBP2 was bound only to the SECIS in 48S pre-initiation and 80S pretranslocation complexes. In the complex where the Sec-tRNA Sec was accommodated to the A site but transpeptidation was blocked, SBP2 bound the ribosome and possibly the SECIS element as well, and the SECIS had flexible contacts with the 60S ribosomal subunit involving several ribosomal proteins. Altogether, our findings led to broadening our understanding about the unique mechanism of selenocysteine incorporation in mammals.

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Preparation of misacylated aminoacyl-tRNAPhe's useful as probes of the ribosomal acceptor site

Cited by 68 publications

References 61 publications

Enzymatic aminoacylation of tRNA with unnatural amino acids

Enzymatic aminoacylation of tRNA with unnatural amino acids

Initiating translation with D-amino acids

A novel insight into the mechanism of mammalian selenoprotein synthesis

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