Structural basis of specific tRNA aminoacylation by a small in vitro selected ribozyme

In modern organisms, protein enzymes are solely responsible for the aminoacylation of transfer RNA. However, the evolution of protein synthesis in the RNA world required RNAs capable of catalysing this reaction. Ribozymes that aminoacylate RNA by using activated amino acids have been discovered through selection in vitro. Flexizyme is a 45-nucleotide ribozyme capable of charging tRNA in trans with various activated l-phenylalanine derivatives. In addition to a more than 105 rate enhancement and more than 104-fold discrimination against some non-cognate amino acids, this ribozyme achieves good regioselectivity: of all the hydroxyl groups of a tRNA, it exclusively aminoacylates the terminal 3′-OH. Here we report the 2.8-Å resolution structure of flexizyme fused to a substrate RNA. Together with randomization of ribozyme core residues and reselection, this structure shows that very few nucleotides are needed for the aminoacylation of specific tRNAs. Although it primarily recognizes tRNA through base-pairing with the CCA terminus of the tRNA molecule, flexizyme makes numerous local interactions to position the acceptor end of tRNA precisely. A comparison of two crystallographically independent flexizyme conformations, only one of which appears capable of binding activated phenylalanine, suggests that this ribozyme may achieve enhanced specificity by coupling active-site folding to tRNA docking. Such a mechanism would be reminiscent of the mutually induced fit of tRNA and protein employed by some aminoacyl-tRNA synthetases to increase specificity.

[1]  Z. Otwinowski,et al.  Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[2]  W. Schamel,et al.  Molecular evolution of transfer RNA from two precursor hairpins: Implications for the origin of protein synthesis , 1995, Journal of Molecular Evolution.

[3]  T. Steitz,et al.  Structure of E. coli glutaminyl-tRNA synthetase complexed with tRNA(Gln) and ATP at 2.8 A resolution. , 1989, Science.

[4]  G. Petsko,et al.  Weakly polar interactions in proteins. , 1988, Advances in protein chemistry.

[5]  R Giegé,et al.  Universal rules and idiosyncratic features in tRNA identity. , 1998, Nucleic acids research.

[6]  Thomas A. Steitz,et al.  RNA tertiary interactions in the large ribosomal subunit: The A-minor motif , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[7]  H. Suga,et al.  A ribozyme exclusively aminoacylates the 3'-hydroxyl group of the tRNA terminal adenosine. , 2001, Journal of the American Chemical Society.

[8]  M. Illangasekare,et al.  Specific, rapid synthesis of Phe-RNA by RNA. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[9]  A. Ferré-D’Amaré,et al.  Crystallization and structure determination of a hepatitis delta virus ribozyme: use of the RNA-binding protein U1A as a crystallization module. , 2000, Journal of molecular biology.

[10]  S. Svärd,et al.  Base pairing between Escherichia coli RNase P RNA and its substrate. , 1994, The EMBO journal.

[11]  M. Illangasekare,et al.  Aminoacyl-RNA synthesis catalyzed by an RNA , 1995, Science.

[12]  Olivier Poch,et al.  Partition of tRNA synthetases into two classes based on mutually exclusive sets of sequence motifs , 1990, Nature.

[13]  Randy J Read,et al.  Electronic Reprint Biological Crystallography Likelihood-enhanced Fast Translation Functions Biological Crystallography Likelihood-enhanced Fast Translation Functions , 2022 .

[14]  H. Murakami,et al.  A versatile tRNA aminoacylation catalyst based on RNA. , 2003, Chemistry & biology.

[15]  A. Serganov,et al.  Structural basis for Diels-Alder ribozyme-catalyzed carbon-carbon bond formation , 2005, Nature Structural &Molecular Biology.

[16]  M Yarus,et al.  A tiny RNA that catalyzes both aminoacyl-RNA and peptidyl-RNA synthesis. , 1999, RNA.

[17]  J. Wong.  Origin of genetically encoded protein synthesis: A model based on selection for RNA peptidation , 2005, Origins of life and evolution of the biosphere.

[18]  A. Ferré-D’Amaré,et al.  Use of cis- and trans-ribozymes to remove 5' and 3' heterogeneities from milligrams of in vitro transcribed RNA. , 1996, Nucleic acids research.

[19]  M. Selmer,et al.  Structure of the 70S Ribosome Complexed with mRNA and tRNA , 2006, Science.

[20]  A. Weiner,et al.  tRNA-like structures tag the 3' ends of genomic RNA molecules for replication: implications for the origin of protein synthesis. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[21]  R Giegé,et al.  An operational RNA code for amino acids and possible relationship to genetic code. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[22]  D. Moras,et al.  Class II aminoacyl transfer RNA synthetases: crystal structure of yeast aspartyl-tRNA synthetase complexed with tRNA(Asp) , 1991, Science.

[23]  A. Ferré-D’Amaré,et al.  Crystallization of the Hairpin Ribozyme , 2004 .

[24]  Y. Bessho,et al.  Ribozyme-catalyzed tRNA aminoacylation , 2000, Nature Structural Biology.

[25]  W. Delano.  The PyMOL Molecular Graphics System , 2002 .

[26]  A. Ferré-D’Amaré,et al.  Structural Basis of glmS Ribozyme Activation by Glucosamine-6-Phosphate , 2006, Science.

[27]  Wolfram Saenger,et al.  Principles of Nucleic Acid Structure , 1983 .

[28]  T. Cech,et al.  A preorganized active site in the crystal structure of the Tetrahymena ribozyme. , 1998, Science.

[29]  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.

[30]  S Cusack,et al.  The crystal structure of the ternary complex of T.thermophilus seryl‐tRNA synthetase with tRNA(Ser) and a seryl‐adenylate analogue reveals a conformational switch in the active site. , 1996, The EMBO journal.

[31]  K. Watanabe,et al.  Concurrent molecular recognition of the amino acid and tRNA by a ribozyme. , 2001, RNA.

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

[33]  L. Orgel.  The origin of polynucleotide-directed protein synthesis , 2007, Journal of Molecular Evolution.

[34]  H. Suga,et al.  Outersphere and innersphere coordinated metal ions in an aminoacyl-tRNA synthetase ribozyme. , 2002, Nucleic acids research.

[35]  Hiroshi Murakami,et al.  A highly flexible tRNA acylation method for non-natural polypeptide synthesis , 2006, Nature Methods.

[36]  H. Murakami,et al.  Designer ribozymes: programming the tRNA specificity into flexizyme. , 2004, Journal of the American Chemical Society.

[37]  J. Perona,et al.  Indirect readout of tRNA for aminoacylation. , 2007, Biochemistry.

[38]  G. Eriani,et al.  The structure of an AspRS—tRNAAsp complex reveals a tRNA‐dependent control mechanism , 2001, The EMBO journal.

[39]  H. Suga,et al.  An in vitro evolved precursor tRNA with aminoacylation activity , 2001, The EMBO journal.