tRNAPro anticodon recognition by Thermus thermophilus prolyl-tRNA synthetase

Abstract

For the synthetases possessing the class IIa anticodon-binding domain (ProRS, ThrRS and GlyRS, with the exception of HisRS), the two anticodon bases 35 and 36 are sufficient to uniquely identify the cognate tRNA (GG for proline, GU for threonine, CC for glycine), because these amino acids occupy full codon groups. The structure of ProRSTT in complex with its cognate tRNA shows that these two bases specifically interact with the enzyme, whereas base 34, which can be any base, is stacked under base 33 and makes no interactions with the synthetase. This is in agreement with biochemical experiments which identify bases 35 and 36 as major tRNA identity elements. In contrast, class IIb synthetases (AspRS, AsnRS and LysRS) have a distinct anticodon-binding domain that specifically recognises all three anticodon bases. This again correlates with the requirements of the genetic code for cognate tRNA identification, as the class IIb amino acids occupy half codon groups.

Evolution of multiple, mutually orthogonal prolyl-tRNA synthetase/tRNA pairs for unnatural amino acid mutagenesis in Escherichia coli

“…These examples suggest that it may be possible to evolve the anticodon-binding pocket of PhProRS to recognize and charge Af-tRNA CUA Pro efficiently. The cocrystal structure of the tRNA Pro /ProRS pair from T. thermophilus, which is homologous to the pair identified above, reveals the molecular basis of anticodon recognition by ProRS (18,19). We found that the consensus sequence of the proline anticodon (GG) interacts extensively with several conserved amino acid residues in the anticodon-binding pocket of ProRS ( Fig.…”

“…However, the results described here attest to the plasticity of the tRNA-aaRS interface and its potential utility for evolving variants with unique properties. Crystal structures of ProRS from T. thermophilus (18,19) and Methanothermobacter thermautotrophicus (29) bound to prolyl-adenylate analogs are available, providing excellent structural bases for the evolution of the analogous PhProRS to encode genetically novel proline analogs and other N-modified UAAs. In addition, the development of mutually orthogonal prolyl pairs further expands our ability to incorporate multiple, distinct UAAs into the same protein, eventually leading to genetically encoded unnatural biopolymers.…”

“…The other two nonsense codons, TAA and TGA, and several frameshift codons also have been used successfully (2,3,7,14,15). In the case of the tRNA Pro /ProRS pair, the anticodon is a major identity element for tRNA recognition by its cognate synthetase (16)(17)(18)(19). The consensus sequence of the proline anticodons (NGG) in tRNA Pro interacts extensively with an array of conserved amino acid residues in the anticodonbinding pocket of ProRS, as evidenced in the tRNA Pro /ProRS cocrystal structure from Thermus thermophilus (18,19).…”

“…In the case of the tRNA Pro /ProRS pair, the anticodon is a major identity element for tRNA recognition by its cognate synthetase (16)(17)(18)(19). The consensus sequence of the proline anticodons (NGG) in tRNA Pro interacts extensively with an array of conserved amino acid residues in the anticodonbinding pocket of ProRS, as evidenced in the tRNA Pro /ProRS cocrystal structure from Thermus thermophilus (18,19). As a consequence, alteration of the anticodon negatively affects the tRNA Pro -ProRS interaction, leading to low or undetectable activity in E. coli.…”

Importance of the Anticodon Sequence in the Aminoacylation of tRNAs by Methionyl-tRNA Synthetase and by Valyl-tRNA Synthetase in an Archaebacterium

“…The crystal structure analysis of several aminoacyl-tRNA synthetase-tRNA complexes from eubacteria and eukarya has provided a substantial amount of information on the molecular details of interactions involving these determinants (1)(2)(3)(4)(5). However, little is known either at the biochemical level or at the structural level on interaction between aminoacyl-tRNA synthetases and tRNA in archaea.…”