Abstract
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 post-transfer editing in ThrRS are subject to modulation by the rate of aminoacyl transfer.
Highlights
The accurate transfer of genetic information in living systems requires multiple mechanisms to enhance and preserve the fidelity of gene expression, 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]
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
Summary
The accurate transfer of genetic information in living systems requires multiple mechanisms to enhance and preserve the fidelity of gene expression, in protein synthesis. 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 105) reflects the ability of ARSs to carefully discriminate among chemically similar standard and nonstandard amino acids [3]. 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. Selection of Editing Pathway in ThrRS in fidelity preservation is not well understood, because they have not been studied by detailed kinetic analysis
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