Structural and mechanistic basis of pre- and posttransfer editing by leucyl-tRNA synthetase.

The aminoacyl-tRNA synthetases link tRNAs with their cognate amino acid. In some cases, their fidelity relies on hydrolytic editing that destroys incorrectly activated amino acids or mischarged tRNAs. We present structures of leucyl-tRNA synthetase complexed with analogs of the distinct pre- and posttransfer editing substrates. The editing active site binds the two different substrates using a single amino acid discriminatory pocket while preserving the same mode of adenine recognition. This suggests a similar mechanism of hydrolysis for both editing substrates that depends on a key, completely conserved aspartic acid, which interacts with the alpha-amino group of the noncognate amino acid and positions both substrates for hydrolysis. Our results demonstrate the economy by which a single active site accommodates two distinct substrates in a proofreading process critical to the fidelity of protein synthesis.

[1]  Maria C. Nagan,et al.  Functional role of the prokaryotic proline-tRNA synthetase insertion domain in amino acid editing. , 2002, Biochemistry.

[2]  U. Englisch,et al.  The proofreading of hydroxy analogues of leucine and isoleucine by leucyl-tRNA synthetases from E. coli and yeast. , 1986, Nucleic acids research.

[3]  H. Ogura,et al.  A convenient synthesis of peptide using oxallates , 1983 .

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

[5]  S. Cusack,et al.  Crystallization and preliminary crystallographic analysis of Thermus thermophilus leucyl-tRNA synthetase and its complexes with leucine and a non-hydrolysable leucyl-adenylate analogue. , 2000, Acta crystallographica. Section D, Biological crystallography.

[6]  K. Nebel,et al.  A novel synthesis of sulfamoyl nucleosides , 1994 .

[7]  H. Ueda,et al.  X-ray crystallographic conformational study of 5'-O-[N-(L-alanyl)-sulfamoyl]adenosine, a substrate analogue for alanyl-tRNA synthetase. , 1991, Biochimica et biophysica acta.

[8]  J. Thevelein,et al.  The cell division cycle gene CDC60 encodes cytosolic leucyl-tRNA synthetase in Saccharomyces cerevisiae. , 1992, Gene.

[9]  G. Grübel,et al.  Crystal structures at 2.5 angstrom resolution of seryl-tRNA synthetase complexed with two analogs of seryl adenylate. , 1994, Science.

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

[11]  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.

[12]  S. Martinis,et al.  Non-standard amino acid recognition by Escherichia coli leucyl-tRNA synthetase. , 1997, Nucleic acids symposium series.

[13]  S Cusack,et al.  The 2 Å crystal structure of leucyl‐tRNA synthetase and its complex with a leucyl‐adenylate analogue , 2000, The EMBO journal.

[14]  S. Martinis,et al.  Rational design to block amino acid editing of a tRNA synthetase. , 2002, Journal of the American Chemical Society.

[15]  E. Goldman,et al.  Editing of errors in selection of amino acids for protein synthesis. , 1992, Microbiological reviews.

[16]  C. Ehresmann,et al.  The Structure of Threonyl-tRNA Synthetase-tRNAThr Complex Enlightens Its Repressor Activity and Reveals an Essential Zinc Ion in the Active Site , 1999, Cell.

[17]  S. Martinis,et al.  Enzymatic aminoacylation of sequence-specific RNA minihelices and hybrid duplexes with methionine. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[18]  D. Lloyd.  The cell division cycle , 1982 .

[19]  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.

[20]  Paul Schimmel,et al.  Mutational separation of two pathways for editing by a class I tRNA synthetase. , 2002, Molecular cell.

[21]  I. Apostol,et al.  Incorporation of Norvaline at Leucine Positions in Recombinant Human Hemoglobin Expressed in Escherichia coli* , 1997, The Journal of Biological Chemistry.

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

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

[24]  S. Blanquet,et al.  Covalent methionylation of escherichia coli methionyl‐tRNA synthethase: Identification of the labeled amino acid residues by matrix‐assisted laser desorption‐ionization mass spectrometry , 1997, Protein science : a publication of the Protein Society.

[25]  A G Leslie,et al.  Biological Crystallography Integration of Macromolecular Diffraction Data , 2022 .

[26]  E. Wang,et al.  Effect of alanine-293 replacement on the activity, ATP binding, and editing of Escherichia coli leucyl-tRNA synthetase. , 2001, Biochemistry.

[27]  P. Schimmel,et al.  Blocking site-to-site translocation of a misactivated amino acid by mutation of a class I tRNA synthetase , 2002, Proceedings of the National Academy of Sciences of the United States of America.

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

[29]  Yi Tang,et al.  Attenuation of the editing activity of the Escherichia coli leucyl-tRNA synthetase allows incorporation of novel amino acids into proteins in vivo. , 2002, Biochemistry.

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

[31]  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.

[32]  U. RajBhandary,et al.  Direct analysis of aminoacylation levels of tRNAs in vivo. Application to studying recognition of Escherichia coli initiator tRNA mutants by glutaminyl-tRNA synthetase. , 1991, The Journal of biological chemistry.

[33]  Collaborative Computational,et al.  The CCP4 suite: programs for protein crystallography. , 1994, Acta crystallographica. Section D, Biological crystallography.

[34]  R. Lerner,et al.  Biochemical characterization and structural analysis of a highly proficient cocaine esterase. , 2002, Biochemistry.

[35]  P. Schimmel,et al.  Plasticity of Recognition of the 3′-End of Mischarged tRNA by Class I Aminoacyl-tRNA Synthetases* , 2002, The Journal of Biological Chemistry.

[36]  A. Ferré-D’Amaré,et al.  Transition State Stabilization by a Catalytic RNA , 2002, Science.

[37]  Y. Mechulam,et al.  Structure of crystalline D-Tyr-tRNA(Tyr) deacylase. A representative of a new class of tRNA-dependent hydrolases. , 2001, The Journal of biological chemistry.

[38]  Y L Wang,et al.  CP1 domain in Escherichia coli leucyl-tRNA synthetase is crucial for its editing function. , 2000, Biochemistry.

[39]  S. Martinis,et al.  Aminoacyl-tRNA synthetases: a new image for a classical family. , 1999, Biochimie.

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

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

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