第二套遗传密码子science_1998
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Insights into Editing from an Ile-tRNA Synthetase Structure with tRNA Ile and Mupirocin Laura F. Silvian, et al. Science 285, 1074 (1999); DOI: 10.1126/science.285.5430.1074
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Insights into Editing from an Ile-tRNA Synthetase Structure
with tRNAIle and Mupirocin
Laura F. Silvian,1,3* Jimin Wang,1 Thomas A. Steitz1,2,3
Isoleucyl–transfer RNA (tRNA) synthetase (IleRS) joins Ile to tRNAIle at its synthetic active site and hydrolyzes incorrectly acylated amino acids at its editing active site. The 2.2 angstrom resolution crystal structure of Staphylococcus aureus IleRS complexed with tRNAIle and Mupirocin shows the acceptor strand of the tRNAIle in the continuously stacked, A-form conformation with the 3Ј terminal nucleotide in the editing active site. To position the 3Ј terminus in the synthetic active site, the acceptor strand must adopt the hairpinned conformation seen in tRNAGln complexed with its synthetase. The amino acid editing activity of the IleRS may result from the incorrect products shuttling between the synthetic and editing active sites, which is reminiscent of the editing mechanism of DNA polymerases.
mechanism involving a tRNA-dependent shuttling of the incorrect product between the two active sites.
The determination of the tRNA-IleRS strucA
B
ture is summarized in Table 1. The IleRS structure can be divided into three regions of differing function (Fig. 1A). The region that recognizes an unusual anticodon loop conformation is formed by three domains at the COOHterminus and one at the extreme NH2-terminus. The dinucleotide (or Rossmann) fold domain forms the core that both activates and transfers the amino acid. The editing domain (connective peptide 1, or CP1) removes any incorrectly incorporated amino acid. tRNAIle binds along the long axis of the cigar-shaped IleRS structure. An antiparallel pair of ␣ helices binds the minor groove of the tRNA acceptor stem; however, nucleotides in the D-loop of tRNAIle that were reported to be important for editing (7) do not interact directly with monomeric IleRS. Interactions between IleRS and tRNAIle that specify recognition of the tRNAIle sequence, as well as interactions with Mupirocin, are discussed elsewhere (9).
Science (print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by the American Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. Copyright 1999 by the American Association for the Advancement of Science; all rights reserved. The title Science is a registered trademark of AAAS.
The aminoacyl-tRNA synthetases that aminoacylate tRNAs with Ile, Thr, and Ala must discriminate against the amino acid containing one less methyl group. Based on thermodynamic considerations, Pauling (1) estimated that isoleucyl-tRNA synthetase (IleRS) could distinguish between Ile and Val by only a factor of 5; however, fewer than 1 error in 3000 occurs (2). To obtain the observed specificity of IleRS, a proofreading activity that hydrolyzes incorrect product is involved (3). It has been proposed (4) that selectivity is achieved by a “double-sieve” mechanism in which amino acids larger than the correct one are sterically excluded at the synthetic active site; in contrast, only smaller amino acids are sterically allowed to bind at a hydrolytic editing active site. The editing and synthetic active sites reside on separate domains (5). In Thermus thermophilus apo-IleRS, they are separated by 34 Å and have different specificities for amino acid binding (6 ).
This copy is for your personal, non-commercial use only.
If you wish to distribute this article to others, you can order high-quality copies for your colleagues, clients, or customers by clicking here. Permission to republish or repurpose articles or portions of articles can be obtained by following the guidelines here. The following resources related to this article are available online at (this infomation is current as of November 15, 2011 ): Updated information and services, including high-resolution figures, can be found in the online version of this article at: /content/285/5430/1074.full.html This article cites 17 articles, 5 of which can be accessed free: /content/285/5430/1074.full.html#ref-list-1 This article has been cited by 200 article(s) on the ISI Web of Science This article has been cited by 85 articles hosted by HighWire Press; see: /content/285/5430/1074.full.html#related-urls This article appears in the following subject collections: Biochemistry /cgi/collection/biochem
We address here how amino acid editing is achieved by an active site that is 34 Å distant from the site where the incorrect product is formed and why editing of either the aminoacyl adenylate or the aminoacyl-tRNA requires tRNAIle (7). One possibility is that the active sites might be brought closer together by a rotation of the editing domain upon addition of tRNA to form a closed cavity that contains the tRNA 3Ј terminus and both active sites (8). However, the crystal structure of S. aureus IleRS complexed with tRNAIle and Mupirocin presented here suggests an alternative
Insights into Editing from an Ile-tRNA Synthetase Structure with tRNA Ile and Mupirocin Laura F. Silvian, et al. Science 285, 1074 (1999); DOI: 10.1126/science.285.5430.1074
Downloaded from on NORTS
Insights into Editing from an Ile-tRNA Synthetase Structure
with tRNAIle and Mupirocin
Laura F. Silvian,1,3* Jimin Wang,1 Thomas A. Steitz1,2,3
Isoleucyl–transfer RNA (tRNA) synthetase (IleRS) joins Ile to tRNAIle at its synthetic active site and hydrolyzes incorrectly acylated amino acids at its editing active site. The 2.2 angstrom resolution crystal structure of Staphylococcus aureus IleRS complexed with tRNAIle and Mupirocin shows the acceptor strand of the tRNAIle in the continuously stacked, A-form conformation with the 3Ј terminal nucleotide in the editing active site. To position the 3Ј terminus in the synthetic active site, the acceptor strand must adopt the hairpinned conformation seen in tRNAGln complexed with its synthetase. The amino acid editing activity of the IleRS may result from the incorrect products shuttling between the synthetic and editing active sites, which is reminiscent of the editing mechanism of DNA polymerases.
mechanism involving a tRNA-dependent shuttling of the incorrect product between the two active sites.
The determination of the tRNA-IleRS strucA
B
ture is summarized in Table 1. The IleRS structure can be divided into three regions of differing function (Fig. 1A). The region that recognizes an unusual anticodon loop conformation is formed by three domains at the COOHterminus and one at the extreme NH2-terminus. The dinucleotide (or Rossmann) fold domain forms the core that both activates and transfers the amino acid. The editing domain (connective peptide 1, or CP1) removes any incorrectly incorporated amino acid. tRNAIle binds along the long axis of the cigar-shaped IleRS structure. An antiparallel pair of ␣ helices binds the minor groove of the tRNA acceptor stem; however, nucleotides in the D-loop of tRNAIle that were reported to be important for editing (7) do not interact directly with monomeric IleRS. Interactions between IleRS and tRNAIle that specify recognition of the tRNAIle sequence, as well as interactions with Mupirocin, are discussed elsewhere (9).
Science (print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by the American Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. Copyright 1999 by the American Association for the Advancement of Science; all rights reserved. The title Science is a registered trademark of AAAS.
The aminoacyl-tRNA synthetases that aminoacylate tRNAs with Ile, Thr, and Ala must discriminate against the amino acid containing one less methyl group. Based on thermodynamic considerations, Pauling (1) estimated that isoleucyl-tRNA synthetase (IleRS) could distinguish between Ile and Val by only a factor of 5; however, fewer than 1 error in 3000 occurs (2). To obtain the observed specificity of IleRS, a proofreading activity that hydrolyzes incorrect product is involved (3). It has been proposed (4) that selectivity is achieved by a “double-sieve” mechanism in which amino acids larger than the correct one are sterically excluded at the synthetic active site; in contrast, only smaller amino acids are sterically allowed to bind at a hydrolytic editing active site. The editing and synthetic active sites reside on separate domains (5). In Thermus thermophilus apo-IleRS, they are separated by 34 Å and have different specificities for amino acid binding (6 ).
This copy is for your personal, non-commercial use only.
If you wish to distribute this article to others, you can order high-quality copies for your colleagues, clients, or customers by clicking here. Permission to republish or repurpose articles or portions of articles can be obtained by following the guidelines here. The following resources related to this article are available online at (this infomation is current as of November 15, 2011 ): Updated information and services, including high-resolution figures, can be found in the online version of this article at: /content/285/5430/1074.full.html This article cites 17 articles, 5 of which can be accessed free: /content/285/5430/1074.full.html#ref-list-1 This article has been cited by 200 article(s) on the ISI Web of Science This article has been cited by 85 articles hosted by HighWire Press; see: /content/285/5430/1074.full.html#related-urls This article appears in the following subject collections: Biochemistry /cgi/collection/biochem
We address here how amino acid editing is achieved by an active site that is 34 Å distant from the site where the incorrect product is formed and why editing of either the aminoacyl adenylate or the aminoacyl-tRNA requires tRNAIle (7). One possibility is that the active sites might be brought closer together by a rotation of the editing domain upon addition of tRNA to form a closed cavity that contains the tRNA 3Ј terminus and both active sites (8). However, the crystal structure of S. aureus IleRS complexed with tRNAIle and Mupirocin presented here suggests an alternative