Changing the Identity of a tRNA by Introducing a G-U Wobble Pair Near the 3′ Acceptor End

McClain, William H.; Foss, K.

doi:10.1126/science.2452483

Cited by 357 publications

(258 citation statements)

References 31 publications

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“…In order to be able to visualize and quantify the level of suppression obtained upon insertion of the Pyl analogues we used a b-galactosidase based reporter system. The E. coli XAC strains in which a UAG in-frame codon has been inserted in the lacI part of a lacI-lacZ fusion gene have been extensively used to assess sequence-function relationships of a wide range of tRNA molecules by quantifying the b-galactosidase activity resulting from suppression of the amber stop codon [20][21][22]. We transformed the E. coli XAC/A24 strain with plasmids carrying pylS and pylT genes.…”

Section: Insertion Of Pyl Analogues Into Proteins In E Colimentioning

confidence: 99%

Pyrrolysine analogues as substrates for pyrrolysyl‐tRNA synthetase

et al. 2006

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In certain methanogenic archaea a new amino acid, pyrrolysine (Pyl), is inserted at in-frame UAG codons in the mRNAs of some methyltransferases. Pyl is directly acylated onto a suppressor tRNA Pyl by pyrrolysyl-tRNA synthetase (PylRS). Due to the lack of a readily available Pyl source, we looked for structural analogues that could be aminoacylated by PylRS onto tRNA Pyl . We report here the in vitro aminoacylation of tRNA Pyl by PylRS with two Pyl analogues: -prolyl-lysine) and N-e-cyclopentyloxycarbonyl-L L-lysine (Cyc). Escherichia coli, transformed with the tRNA Pyl and PylRS genes, suppressed a lacZ amber mutant dependent on the presence of D D-prolyl-lysine or Cyc in the medium, implying that the E. coli translation machinery is able to use Cyc-tRNA Pyl and D D-prolyl-lysine-tRNA Pyl as substrates during protein synthesis. Furthermore, the formation of active b-galactosidase shows that a specialized mRNA motif is not essential for stop-codon recoding, unlike for selenocysteine incorporation.

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Section: Insertion Of Pyl Analogues Into Proteins In E Colimentioning

confidence: 99%

Pyrrolysine analogues as substrates for pyrrolysyl‐tRNA synthetase

et al. 2006

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“…For tRNA Ala , the major determinant for aminoacylation is a G3:U70 base pair in the acceptor stem (32,33). Transfer of this base pair into other tRNA confers aminoacylation with alanine (32)(33)(34).…”

Section: Strategy For Identifying Role Of Trna Structure In Editingmentioning

confidence: 99%

“…Transfer of this base pair into other tRNA confers aminoacylation with alanine (32)(33)(34). In addition, the 12-bp minihelix domain of tRNA Ala (Fig.…”

Section: Strategy For Identifying Role Of Trna Structure In Editingmentioning

confidence: 99%

Structure-specific tRNA Determinants for Editing a Mischarged Amino Acid

Beebe¹,

Merriman

Schimmel

2003

Journal of Biological Chemistry

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Alanyl-tRNA synthetase efficiently aminoacylates tRNA Ala and an RNA minihelix that comprises just one domain of the two-domain L-shaped tRNA structure. It also clears mischarged tRNA Ala using a specialized domain in its C-terminal half. In contrast to full-length tRNA Ala , minihelix Ala was robustly mischarged and could not be edited. Addition in trans of the missing anticodon-containing domain did not activate editing of mischarged minihelix Ala . To understand these differences between minihelix Ala and tRNA Ala , several chimeric full tRNAs were constructed. These had the acceptor stem of a non-cognate tRNA replaced with the stem of tRNA Ala . The chimeric tRNAs collectively introduced multiple sequence changes in all parts but the acceptor stem. However, although the acceptor stem in isolation (as the minihelix) lacked determinants for editing, alanyl-tRNA synthetase effectively cleared a mischarged amino acid from each chimeric tRNA. Thus, a covalently continuous two-domain structure per se, not sequence, is a major determinant for clearance of errors of aminoacylation by alanyl-tRNA synthetase. Because errors of aminoacylation are known to be deleterious to cell growth, structure-specific determinants constitute a powerful selective pressure to retain the format of the two-domain L-shaped tRNA.

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“…Escherichia coli AlaRS was among the first aaRSs that were cloned, sequenced, and characterized genetically and biochemically (1,5,6). tRNA Ala conserves a unique G3:U70 wobble base pair in the acceptor stem, and this base pair dictates the tRNA identity toward AlaRS (7,8). This remarkable finding, that a small number of nucleotide residues serve as the predominant determinant for the tRNA identity, accelerated the search for the identity determinants of other aaRS-tRNA pairs.…”

mentioning

confidence: 99%

Unique protein architecture of alanyl-tRNA synthetase for aminoacylation, editing, and dimerization

Naganuma

Sekine

Fukunaga

et al. 2009

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

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Alanyl-tRNA synthetase (AlaRS) specifically recognizes the major identity determinant, the G3:U70 base pair, in the acceptor stem of tRNA Ala by both the tRNA-recognition and editing domains. In this study, we solved the crystal structures of 2 halves of Archaeoglobus fulgidus AlaRS: AlaRS-⌬C, comprising the aminoacylation, tRNA-recognition, and editing domains, and AlaRS-C, comprising the dimerization domain. The aminoacylation/tRNA-recognition domains contain an insertion incompatible with the class-specific tRNA-binding mode. The editing domain is fixed tightly via hydrophobic interactions to the aminoacylation/tRNA-recognition domains, on the side opposite from that in threonyl-tRNA synthetase. A groove formed between the aminoacylation/tRNA-recognition domains and the editing domain appears to be an alternative tRNA-binding site, which might be used for the aminoacylation and/or editing reactions. Actually, the amino acid residues required for the G3:U70 recognition are mapped in this groove. The dimerization domain consists of helical and globular subdomains. The helical subdomain mediates dimerization by forming a helixloop-helix zipper. The globular subdomain, which is important for the aminoacylation and editing activities, has a positively-charged face suitable for tRNA binding.crystal structure ͉ dimerization domain ͉ aminoacyl-tRNA synthetase ͉ proofreading ͉ wobble base pair

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Changing the Identity of a tRNA by Introducing a G-U Wobble Pair Near the 3′ Acceptor End

Cited by 357 publications

References 31 publications

Pyrrolysine analogues as substrates for pyrrolysyl‐tRNA synthetase

Pyrrolysine analogues as substrates for pyrrolysyl‐tRNA synthetase

Structure-specific tRNA Determinants for Editing a Mischarged Amino Acid

Unique protein architecture of alanyl-tRNA synthetase for aminoacylation, editing, and dimerization

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