Amino Acid Discrimination by a Class I Aminoacyl-tRNA Synthetase Specified by Negative Determinants

Bullock, T.L.; Uter, Nathan T.; Nissan, Tracy; Perona, John J.

doi:10.1016/s0022-2836(03)00305-x

Cited by 98 publications

(153 citation statements)

References 45 publications

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“…Aminoacylated tRNA (as 3Ј-aminoacylated A76) and nonreacted substrate (as unmodified AMP) were separated by TLC and quantitated by using phosphorimaging analysis, as described in ref. 12.…”

Section: Methodsmentioning

confidence: 99%

“…Further, comparisons of the unliganded and tRNA-bound GlnRS structures show that tRNA binding drives an extensive set of conformational changes through all four domains of the enzyme (9,10). The conformational rearrangements converge in the active-site domain, where portions of both the amino acidand ATP-binding sites are misoriented with respect to each other in the absence of tRNA (9,11,12). Because the tRNA also is rearranged compared with its presumed solution structure, formation of the GlnRS-tRNA complex is best described as a mutual induced fit involving conformational changes of both macromolecular partners.…”

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confidence: 99%

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Long-range intramolecular signaling in a tRNA synthetase complex revealed by pre-steady-state kinetics

Uter

Perona

2004

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

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107

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Pre-steady-state kinetic studies of Escherichia coli glutaminyl-tRNA synthetase conclusively demonstrate the existence of long-distance pathways of communication through the protein-RNA complex. Measurements of aminoacyl-tRNA synthesis reveal a rapid burst of product formation followed by a slower linear increase corresponding to k cat. Thus, a step after chemistry but before regeneration of active enzyme is rate-limiting for synthesis of Gln-tRNA Gln . Single-turnover kinetics validates these observations, confirming that the rate of the chemical step for tRNA aminoacylation (k chem) exceeds the steady-state rate by nearly 10-fold. The concentration dependence of the single-turnover reaction further reveals that the glutamine Kd is significantly higher than the steady-state K m value. The separation of binding from catalytic events by transient kinetics now allows precise interpretation of how alterations in tRNA structure affect the aminoacylation reaction. Mutation of U35 in the tRNA anticodon loop decreases k chem by 30-fold and weakens glutamine binding affinity by 20-fold, demonstrating that the active-site configuration depends on enzyme-tRNA contacts some 40 Å distant. By contrast, mutation of the adjacent G36 has very small effects on k chem and Kd for glutamine. Together with x-ray crystallographic data, these findings allow a comparative evaluation of alternative long-range signaling pathways and lay the groundwork for systematic exploration of how induced-fit conformational transitions may control substrate selection in this model enzyme-RNA complex.R ecognition between proteins and RNA generally occurs by an induced-fit mechanism, in which the protein, the RNA, or both undergo conformational changes en route to the final bound complex (1, 2). Although induced fit necessarily incurs an entropic penalty compared with a preformed and rigid interaction, the built-in flexibility nonetheless may help to increase the kinetic association rate, thus lowering the free-energy barrier for complex formation. In enzymatic reactions, it is also established that induced fit can provide a means to increase substrate specificity: Noncognate substrates may induce partial or incorrect rearrangements, leading to misalignment of reactive moieties along the pathway to the transition state (3). Thus, enzymes that modify RNA may employ induced fit for substrate discrimination at both binding and catalytic steps of the reaction.Aminoacyl-tRNA synthetases are superb model systems for investigating the operation of induced fit in enzyme-RNA complexes. In all organisms, these enzymes maintain fidelity of the genetic code by catalyzing the synthesis of cognate aminoacyl-tRNAs for use in protein synthesis (4). This synthesis occurs by means of a two-step reaction in which the amino acid is first activated to form an aminoacyl adenylate intermediate with release of pyrophosphate (PP i ). In the second step, the nucleophilic oxygen from the 2Ј-OH or 3Ј-OH group on the 3Ј-terminal tRNA ribose sugar attacks the mixed anhydride interm...

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Long-range intramolecular signaling in a tRNA synthetase complex revealed by pre-steady-state kinetics

Uter

Perona

2004

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

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107

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“…Samples with only unlabelled tRNA Asn were aminoacylated in parallel and were used in the glutaminase assays. In place of checking the levels of aminoacylation as described elsewhere (Bullock et al, 2003), we checked the level of Asn-tRNA Asn formation by measuring the amount of Asn formed in the transamidation reaction (see below) in the presence of a high concentration (4 nM) of GatCAB wt .…”

Section: Figmentioning

confidence: 99%

Natural insertion of the bro-1 β-lactamase gene into the gatCAB operon affects Moraxella catarrhalis aspartyl-tRNAAsn amidotransferase activity

2012

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Only about half of bacterial species use an asparaginyl-tRNA synthetase (AsnRS) to attach Asn to its cognate tRNA Asn . Other bacteria, including the human pathogen Moraxella catarrhalis, a causative agent of otitis media, lack a gene encoding AsnRS, and form Asn-tRNA Asn by an indirect pathway catalysed by two enzymes: first, a non-discriminating aspartyl-tRNA synthetase (ND-AspRS) catalyses the formation of aspartyl-tRNA Asn (Asp-tRNA Asn ); then, a tRNAdependent amidotransferase (GatCAB) transamidates this 'incorrect' product into Asn-tRNA Asn . As M. catarrhalis has a Gln-tRNA synthetase, its GatCAB functions as an Asp-tRNA Asn amidotransferase. This pathogen rapidly evolved to about 90 % ampicillin resistance worldwide by insertion of a bro-1 b-lactamase gene within the gatCAB operon. Comparison of the GatCAB subunits from bro-1 b-lactamase-positive and bro-negative strains showed that the laterally transferred bro-1 gene, inserted into the gatCAB operon, affected the C-terminal sequence of GatA. The identity between the C-terminal sequences of GatA wt (residues 479-491) and of GatA BRO-1 (residues 479-492) was about 36 %, whereas the rest of the GatA sequence was relatively conserved. The characterization of these two distinct GatCABs as well as the hybrid GatCAB containing GatA(1-478) wt (479-492) BRO-1 and truncated GatCAB enzymes of M. catarrhalis showed that the substitution in GatA wt of residues 479-492 of GatA BRO-1 causes increased specificity for glutamine, and decreased specificity for Asp-tRNA Asn in the transamidation reaction. We conclude that the bro gene insertion has altered the kinetic parameters of Asp-tRNA Asn amidotransferase, and we propose a model for gatA evolution after the insertion of bro-1 at the carboxyl end of gatA.

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“…Reactions were stopped by removing 1 l of the reaction and adding it to 3 l of 2.5 units/ l nuclease P1 (where 1 unit liberates 4 moles of orthophosphate from 3ЈAMP per minute at 37°C; American Bioanalytical, Natick, MA) and incubated at 25°C for 30 min. Nuclease P1 digests were spotted on polyethyleneimine-cellulose plates (Baker, Phillipsburg, NJ), and [␣-32 P]AMP (from uncharged tRNA) and aminoacyl-[␣-32 P]AMP (from charged tRNA) were separated in a running buffer of 0.1 M sodium acetate and 5% acetic acid (31) and visualized by using a phosphoimager system. Kinetic parameters represent the results of at least three independent experiments.…”

Section: Purification Of Recombinant M Barkeri Pylrs and In Vitro Trmentioning

confidence: 99%

Pyrrolysine is not hardwired for cotranslational insertion at UAG codons

Ambrogelly¹,

Gundllapalli²,

Herring³

et al. 2007

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

121

126

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Pyrrolysine (Pyl), the 22nd naturally encoded amino acid, gets acylated to its distinctive UAG suppressor tRNA Pyl by the cognate pyrrolysyl-tRNA synthetase (PylRS). Here we determine the RNA elements required for recognition and aminoacylation of tRNA Pyl in vivo by using the Pyl analog N--cyclopentyloxycarbonyl-L-lysine. Forty-two Methanosarcina barkeri tRNA Pyl variants were tested in Escherichia coli for suppression of the lac amber A24 mutation; then relevant tRNA Pyl mutants were selected to determine in vivo binding to M. barkeri PylRS in a yeast three-hybrid system and to measure in vitro tRNA Pyl aminoacylation. tRNA Pyl identity elements include the discriminator base, the first base pair of the acceptor stem, the T-stem base pair G51:C63, and the anticodon flanking nucleotides U33 and A37. Transplantation of the tRNA Pyl identity elements into the mitochondrial bovine tRNA Ser scaffold yielded chimeric tRNAs active both in vitro and in vivo. Because the anticodon is not important for PylRS recognition, a tRNA Pyl variant could be constructed that efficiently suppressed the lac opal U4 mutation in E. coli. These data suggest that tRNA Pyl variants may decode numerous codons and that tRNA Pyl :PylRS is a fine orthogonal tRNA:synthetase pair that facilitated the late addition of Pyl to the genetic code.orthogonal tRNA ͉ suppression ͉ tRNA identity ͉ pyrrolysyl-tRNA synthetase ͉ aminoacyl-tRNA synthetase

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Amino Acid Discrimination by a Class I Aminoacyl-tRNA Synthetase Specified by Negative Determinants

Cited by 98 publications

References 45 publications

Long-range intramolecular signaling in a tRNA synthetase complex revealed by pre-steady-state kinetics

Long-range intramolecular signaling in a tRNA synthetase complex revealed by pre-steady-state kinetics

Natural insertion of the bro-1 β-lactamase gene into the gatCAB operon affects Moraxella catarrhalis aspartyl-tRNAAsn amidotransferase activity

Pyrrolysine is not hardwired for cotranslational insertion at UAG codons

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