RNA-assisted catalysis in a protein enzyme: The 2′-hydroxyl of tRNA
            <sup>Thr</sup>
            A76 promotes aminoacylation by threonyl-tRNA synthetase

121

Background: Error-prone aminoacyl-tRNA synthetases clear noncognate aminoacyl-adenylates and misacylated tRNAs within synthetic and editing sites, respectively. Results: Product release limits the rate of post-transfer editing by leucyl-tRNA synthetase. Conclusion: Kinetic partitioning of misacylated tRNA determines the relative contribution of cis and trans editing. Significance: In contrast to DNA polymerases, error correction in class I tRNA synthetases relies on substrate selection by the editing site.

Section: Leurs Resembles Valrs In Utilizing Post-transfer Editing As mentioning

confidence: 99%

Kinetic Partitioning between Synthetic and Editing Pathways in Class I Aminoacyl-tRNA Synthetases Occurs at Both Pre-transfer and Post-transfer Hydrolytic Steps

Cvetešić

Perona

Gruić-Sovulj

2012

121

“…The resulting plots were fit to a complex equation featuring a bi-exponential term followed by a linear phase, as described under "Data Analysis." Single turnover aminoacyl transfer experiments employing the post-transfer editing deficient H73A ThrRS were performed as described previously (34). Briefly, the pre-formed ThrRS-[…”

Section: Methodsmentioning

confidence: 99%

“…The purification of ⌬N1N2 ThrRS included 10% glycerol throughout the procedure. The fully modified version of tRNA Thr was prepared from an Escherichia coli overexpression strain as described previously (34).…”

Section: Methodsmentioning

confidence: 99%

“…Preparation of Enzymes and tRNA-ThrRS and CCA adding enzymes were expressed and purified as described previously (34), employing nickel-nitrilotriacetic acid affinity chromatography. Active fractions were identified by SDS-PAGE, pooled, concentrated by low speed centrifugation through Centricon concentrators, and then dialyzed against buffer A (10 mM Tris, pH 8.0, 100 mM KCl, 10 mM MgCl 2 , 3 mM ␤-mercaptoethanol).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Aminoacyl Transfer Rate Dictates Choice of Editing Pathway in Threonyl-tRNA Synthetase

Minajigi

Francklyn

2010

Self Cite

Aminoacyl-tRNA synthetases hydrolyze aminoacyl adenylates and aminoacyl-tRNAs formed from near-cognate amino acids, thereby increasing translational fidelity. The contributions of pre-and post-transfer editing pathways to the fidelity of Escherichia coli threonyl-tRNA synthetase (ThrRS) were investigated by rapid kinetics. In the pre-steady state, asymmetric activation of cognate threonine and noncognate serine was observed in the active sites of dimeric ThrRS, with similar rates of activation. In the absence of tRNA, seryl-adenylate was hydrolyzed 29-fold faster by the ThrRS catalytic domain than threonyl-adenylate. The rate of seryl transfer to cognate tRNA was only 2-fold slower than threonine. Experiments comparing the rate of ATP consumption to the rate of aminoacyl-tRNA AA formation demonstrated that pre-transfer hydrolysis contributes to proofreading only when the rate of transfer is slowed significantly. Thus, the relative contributions of pre-and posttransfer editing in ThrRS are subject to modulation by the rate of aminoacyl transfer.The accurate transfer of genetic information in living systems requires multiple mechanisms to enhance and preserve the fidelity of gene expression, particularly in protein synthesis. The 1-3 errors per 10,000 amino acids incorporated during protein synthesis largely originate from errors in decoding (1) but can arise from previous steps, including the aminoacylation reaction catalyzed by aminoacyl-tRNA synthetases (ARSs) 2 (2). In the first of two half-reactions, ARSs condense amino acid and ATP to form a noncovalently enzyme-bound adenylate, followed by a transfer reaction that produces aminoacyl-tRNA and AMP as products. This highly accurate reaction (on the order of 1 error in 10 5 ) reflects the ability of ARSs to carefully discriminate among chemically similar standard and nonstandard amino acids (3). Mutations in ARSs or their cognate tRNAs can nonetheless create errors associated with significant molecular pathologies, particularly in neural tissues (4).Amino acids that differ by a single methyl group pose a particular discrimination challenge for ARSs (3,5,6). An additional methyl group on an amino acid typically provides no more than ϳ1 kcal/mol of incremental binding energy, imposing an upper limit of discrimination on the order of 1 in 5 (7). To account for the enhanced discrimination seen in living systems, a "double sieve" model was proposed (8). Amino acids larger than cognate are excluded by the "coarse sieve" of the aminoacylation site, whereas smaller or isosteric amino acids are cleaved from the end of the tRNA by the "fine sieve" of the editing site. In principle, noncognate amino acids can be edited both by increased hydrolysis of the adenylate (pre-transfer editing) or by hydrolysis of the mis-acylated tRNA (post-transfer editing). Either or both reactions can formally occur in a dedicated editing site structurally distinct from the standard synthetic site. Because of differences between systems, and the absence of a comprehensive kinetic description ...

“…It is now evident that the essential difference between the two enzymes is the extremely fast transfer rate constant in ValRS, as compared with the slow transfer in IleRS. Comparisons with the transfer rates in other tRNA synthetases show that the behavior of IleRS is most unusual, because fast tRNA transfer is commonly observed among AARS whether or not they also possess editing activities (44,47,48).…”

Section: Kinetic Partitioning Of Aa-amp Within the Synthetic Site Detmentioning

confidence: 99%

Partitioning of tRNA-dependent Editing between Pre- and Post-transfer Pathways in Class I Aminoacyl-tRNA Synthetases

Dulić

Cvetešić

Perona

et al. 2010

175

Hydrolytic editing activities are present in aminoacyl-tRNA synthetases possessing reduced amino acid discrimination in the synthetic reactions. Post-transfer hydrolysis of misacylated tRNA in class I editing enzymes occurs in a spatially separate domain inserted into the catalytic Rossmann fold, but the location and mechanisms of pre-transfer hydrolysis of misactivated amino acids have been uncertain. Here, we use novel kinetic approaches to distinguish among three models for pre-transfer editing by Escherichia coli isoleucyl-tRNA synthetase (IleRS). We demonstrate that tRNA-dependent hydrolysis of noncognate valyl-adenylate by IleRS is largely insensitive to mutations in the editing domain of the enzyme and that noncatalytic hydrolysis after release is too slow to account for the observed rate of clearing. Measurements of the microscopic rate constants for amino acid transfer to tRNA in IleRS and the related valyl-tRNA synthetase (ValRS) further suggest that pre-transfer editing in IleRS is an enzyme-catalyzed activity residing in the synthetic active site. In this model, the balance between pretransfer and post-transfer editing pathways is controlled by kinetic partitioning of the noncognate aminoacyl-adenylate. Rate constants for hydrolysis and transfer of a noncognate intermediate are roughly equal in IleRS, whereas in ValRS transfer to tRNA is 200-fold faster than hydrolysis. In consequence, editing by ValRS occurs nearly exclusively by post-transfer hydrolysis in the editing domain, whereas in IleRS both pre-and post-transfer editing are important. In both enzymes, the rates of amino acid transfer to tRNA are similar for cognate and noncognate aminoacyl-adenylates, providing a significant contrast with editing DNA polymerases.