Editing mechanisms in aminoacylation of tRNA: ATP consumption and the binding of aminoacyl-tRNA by elongation factor Tu

Mulvey, R.S.; Fersht, A.R.

Biochemistry 16(21): 4731-4737


ISSN/ISBN: 0006-2960
PMID: 242929
DOI: 10.1021/bi00640a031
Accession: 068525061

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Stoichiometry of the hydrolysis of ATP and aminoacylation of tRNA was examined to determine whether the editing mechanism that is used to correct errors in the recognition of the amino acid also leads to the wasteful hydrolysis of ATP in the biologically correct reactions. Inhibition of the isoleucyl-tRNA synthetase catalyzed deacylation of Val-tRNAIle by binding to the elongation factor Tu-GTP complex was examined to see if this could prevent editing. A systematic survey of 9 aminocyl-tRNA synthetases, including the isoleucyl-tRNA synthetase under a wide range of conditions, shows that at least 0.95 mol of aminoacyl-tRNA is formed for every mol of ATP hydrolyzed. This is considerably higher than the stoichiometry of 0.66 .+-. 0.2 recently found for the isoleucyl-tRNA synthetase. Reasons are suggested for the discrepancy between the 2 results. The only major waste of ATP hydrolysis appears to occur as a direct result of the activation of valine by the isoleucyl-tRNA synthetase followed by editing. This is calculated to occur in vivo with a frequency of 1 mol of valine activated per 16 mol of isoleucine. Binding of Val-tRNAIle by elongation factor Tu-GTP is found to decrease the rate of the isoleucyl-tRNA synthetase catalyzed deacylation by a factor of about 104. The turnover number for the hydrolysis, about 0.01 s-1, is 100 times slower than the rate of turnover of aminoacyl-tRNA during protein synthesis. If there is an excess of elongation factor Tu in the cell, the amino acid from any misacylated tRNA released from the aminoacyl-tRNA synthetase will be incorporated into proteins. The current status of various postulated editing mechanisms is discussed in light of the above results. [Various aminoacyl tRNA synthetases were isolated from Escherichia coli, Bacillus stearothermophilus and yeast.].