Mutational separation of two pathways for editing by a class I tRNA synthetase.

Aminoacyl tRNA synthetases (aaRSs) catalyze the first step in protein biosynthesis, establishing a connection between codons and amino acids. To maintain accuracy, aaRSs have evolved a second active site that eliminates noncognate amino acids. Isoleucyl tRNA synthetase edits valine by two tRNA(Ile)-dependent pathways: hydrolysis of valyl adenylate (Val-AMP, pretransfer editing) and hydrolysis of mischarged Val-tRNA(Ile) (posttransfer editing). Not understood is how a single editing site processes two distinct substrates--an adenylate and an aminoacyl tRNA ester. We report here distinct mutations within the center for editing that alter adenylate but not aminoacyl ester hydrolysis, and vice versa. These results are consistent with a molecular model that shows that the single editing active site contains two valyl binding pockets, one specific for each substrate.

[1]  P. Schimmel,et al.  Errors from selective disruption of the editing center in a tRNA synthetase. , 2000, Biochemistry.

[2]  A. N. Baldwin,et al.  Transfer ribonucleic acid-induced hydrolysis of valyladenylate bound to isoleucyl ribonucleic acid synthetase. , 1966, The Journal of biological chemistry.

[3]  T. Steitz,et al.  Insights into editing from an ile-tRNA synthetase structure with tRNAile and mupirocin. , 1999 .

[4]  M. Billeter,et al.  MOLMOL: a program for display and analysis of macromolecular structures. , 1996, Journal of molecular graphics.

[5]  Jeffrey H. Miller.  Experiments in molecular genetics , 1972 .

[6]  P. Schimmel,et al.  Aminoacyl tRNA synthetases: general scheme of structure-function relationships in the polypeptides and recognition of transfer RNAs. , 1987, Annual review of biochemistry.

[7]  P. Schimmel,et al.  Residues in a class I tRNA synthetase which determine selectivity of amino acid recognition in the context of tRNA. , 1995, Biochemistry.

[8]  R. B. Loftfield.  THE FREQUENCY OF ERRORS IN PROTEIN BIOSYNTHESIS. , 1963, The Biochemical journal.

[9]  P. Schimmel,et al.  RNA binding determinant in some class I tRNA synthetases identified by alignment-guided mutagenesis. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[10]  F. Cramer,et al.  Isoleucyl-tRNA synthetase from Baker's yeast. Catalytic mechanism, 2',3'-specificity and fidelity in aminoacylation of tRNAIle with isoleucine and valine investigated with initial-rate kinetics using analogs of tRNA, ATP and amino acids. , 1983, European journal of biochemistry.

[11]  A. Fersht,et al.  Editing mechanisms in protein synthesis. Rejection of valine by the isoleucyl-tRNA synthetase. , 1977, Biochemistry.

[12]  D. Belin,et al.  Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter , 1995, Journal of bacteriology.

[13]  S. Martinis,et al.  A conserved threonine within Escherichia coli leucyl-tRNA synthetase prevents hydrolytic editing of leucyl-tRNALeu. , 2001, Biochemistry.

[14]  J. Zou,et al.  Improved methods for building protein models in electron density maps and the location of errors in these models. , 1991, Acta crystallographica. Section A, Foundations of crystallography.

[15]  P. Schimmel,et al.  C-terminal zinc-containing peptide required for RNA recognition by a class I tRNA synthetase. , 1996, Biochemistry.

[16]  P. Marlière,et al.  Enlarging the Amino Acid Set of Escherichia coli by Infiltration of the Valine Coding Pathway , 2001, Science.

[17]  G N Cohen,et al.  Directed evolution of biosynthetic pathways. Recruitment of cysteine thioethers for constructing the cell wall of Escherichia coli. , 1993, The Journal of biological chemistry.

[18]  T. Webster,et al.  Specific sequence homology and three-dimensional structure of an aminoacyl transfer RNA synthetase. , 1984, Science.

[19]  P. Schimmel,et al.  Mutational isolation of a sieve for editing in a transfer RNA synthetase. , 1994, Science.

[20]  R J Read,et al.  Crystallography & NMR system: A new software suite for macromolecular structure determination. , 1998, Acta crystallographica. Section D, Biological crystallography.

[21]  Pascale Romby,et al.  Transfer RNA–Mediated Editing in Threonyl-tRNA Synthetase The Class II Solution to the Double Discrimination Problem , 2000, Cell.

[22]  P. Schimmel,et al.  Aminoacylation error correction , 1996, Nature.

[23]  K. Musier-Forsyth,et al.  Role of zinc ion in translational accuracy becomes crystal clear. , 2000 .

[24]  P. Schimmel,et al.  DNA APTAMER TARGETS TRANSLATIONAL EDITING MOTIF IN A TRNA SYNTHETASE , 1997 .

[25]  F. Cramer,et al.  Isoleucyl-tRNA synthetase from baker's yeast and from Escherichia coli MRE 600. Discrimination of 20 amino acids in aminoacylation of tRNA(Ile)-C-C-A. , 1987, European journal of biochemistry.

[26]  A. Fersht.  Structure and mechanism in protein science , 1998 .

[27]  T. Steitz,et al.  Structural basis for the 3′‐5′ exonuclease activity of Escherichia coli DNA polymerase I: a two metal ion mechanism. , 1991, The EMBO journal.

[28]  D G Vassylyev,et al.  Enzyme structure with two catalytic sites for double-sieve selection of substrate. , 1998, Science.

[29]  R. Starzyk,et al.  Evidence for dispensable sequences inserted into a nucleotide fold. , 1987, Science.

[30]  P. Schimmel,et al.  Rapid deacylation by isoleucyl transfer ribonucleic acid synthetase of isoleucine-specific transfer ribonucleic acid aminoacylated with valine. , 1972, The Journal of biological chemistry.

[31]  Shigeyuki Yokoyama,et al.  Structural Basis for Double-Sieve Discrimination of L-Valine from L-Isoleucine and L-Threonine by the Complex of tRNAVal and Valyl-tRNA Synthetase , 2000, Cell.

[32]  A. Fersht,et al.  Evidence for the double-sieve editing mechanism in protein synthesis. Steric exclusion of isoleucine by valyl-tRNA synthetases. , 1979, Biochemistry.

[33]  P. Schimmel,et al.  Transfer RNA-dependent translocation of misactivated amino acids to prevent errors in protein synthesis. , 1999, Molecular cell.

[34]  K. Musier-Forsyth,et al.  Hydrolytic editing by a class II aminoacyl-tRNA synthetase. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[35]  L. McLaughlin,et al.  Synthesis of the analogue nucleoside 3-deaza-2′-deoxycytidine and its template activity with DNA polymerase , 1999 .

[36]  P. Schimmel,et al.  Functional assembly of a randomly cleaved protein. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[37]  M. Bovee,et al.  Zinc ion mediated amino acid discrimination by threonyl-tRNA synthetase , 2000, Nature Structural Biology.