An essential residue in the flexible peptide linking the two idiosynchratic domains of bacterial tyrosyl-tRNA synthetases

ABSTRACT: Tyrosyl-tRNA synthetase (TyrRS) from Bacillus stearothermophilus comprises three sequential domains: an N-terminal catalytic domain, an R-helical domain with unknown function, and a C-terminal tRNA binding domain (residues 320-419). The properties of the polypeptide segment that links the R-helical and C-terminal domains, were analyzed by measuring the effects of sequence changes on the aminoacylation of tRNATyr with tyrosine. Mutations F323A (Phe323 into Ala), S324A, and G325A showed that the side chain of Phe323 was essential but not those of Ser324 and Gly325. Insertions of Gly residues between Leu322 and Phe323 and the point mutation L322P showed that the position and precise orientation of Phe323 relative to the R-helical domain were important. Insertions of Gly residues between Gly325 and Asp326 and deletion of residues 330-339 showed that the length and flexibility of the sequence downstream from Gly325 were unimportant but that this sequence could not be deleted. Mutations F323A,-L,-Y, and-W showed that the essential property of Phe323 was its aromaticity. The Phe323 side chain contributed to the stability of the initial complex between TyrRS and tRNATyr for 2.0 ( 0.2 kcalâmol-1 and to the stability of their transition state complex for 4.2 ( 0.1 kcalâmol-1, even though it is located far from the catalytic site. The results indicate that the disorder of the C-terminal domain in the crystals of TyrRS is due to the flexibility of the peptide that links it to the helical domain. They identified Phe323 as an essential residue for the recognition of tRNATyr