Structural basis for orthogonal tRNA specificities of tyrosyl-tRNA synthetases for genetic code expansion

Abstract

The archaeal/eukaryotic tyrosyl-tRNA synthetase (TyrRS)-tRNA(Tyr) pairs do not cross-react with their bacterial counterparts. This 'orthogonal' condition is essential for using the archaeal pair to expand the bacterial genetic code. In this study, the structure of the Methanococcus jannaschii TyrRS-tRNA(Tyr)-L-tyrosine complex, solved at a resolution of 1.95 A, reveals that this archaeal TyrRS strictly recognizes the C1-G72 base pair, whereas the bacterial TyrRS recognizes the G1-C72 in a different manner using different residues. These diverse tRNA recognition modes form the basis for the orthogonality. The common tRNA(Tyr) identity determinants (the discriminator, A73 and the anticodon residues) are also recognized in manners different from those of the bacterial TyrRS. Based on this finding, we created a mutant TyrRS that aminoacylates the amber suppressor tRNA with C34 65 times more efficiently than does the wild-type enzyme.

Structural plasticity of an aminoacyl-tRNA synthetase active site

“…However, the D158P and Y32L mutations in the p-BrPhe TyrRS enlarge the binding site to accommodate p-BrPhe and remove the two H-bonds to the hydroxyl oxygen of Tyr that are present in the WT structure ( Fig. 2) (16). The D158P mutation also terminates the ␣ 8 -helix, resulting in formation of a short 3 10 -helix consisting of residues 157-161.…”

“…The final models for both structures had no residues in disallowed regions of the Ramachandran plot as well as a strong difference electron density for the bound amino acid. Like the WT Mj TyrRS (15,16), the NpAla and p-BrPhe Mj synthetases are divided into five regions (the Rossmann-fold catalytic domain; the short N-terminal region; the connective polypeptide 1 domain, which forms the dimer interface; the C-terminal domain; and the KMSKS loop, which links the Rossmann-fold domain to the C-terminal domain). Both mutant synthetases superimpose well with the WT TyrRS-Tyr complex in all regions except the active site (rms deviations were as follows: WT͞pBrPhe, 0.83 Å, over 299 aligned C ␣ atoms; and WT͞NpAla, 0.77 Å, over 294 aligned C ␣ atoms), where the mutations in the p-BrPhe TyrRS and NpAla TyrRS significantly reconfigure the Tyr-binding pocket to selectively bind p-BrPhe or NpAla, respectively (Fig.…”

Residues Lys-149 and Glu-153 Switch the Aminoacylation of tRNATrp in Bacillus subtilis

“…There is little enzymatic data available to support the suggestion that TrpRS contains the corresponding residues that recognize the identity elements in tRNA Trp . However, this suggestion is supported and promoted by the recently disclosed crystal structure of the TyrRS-tRNA complexes (11)(12)(13)(14).…”

“…This implies that the binding of the acceptor-stem in human TrpRS is different to that in B. subtilis TrpRS; however, this barrier might preferentially be removed by the conformational change of human TrpRS in tRNA Trp aminoacylation. tRNA is also a positive substrate for aminoacyl-tRNA synthetase to fold into an active conformation (14,37). The ␣9 helix in human TrpRS has been confirmed to be crucial for tRNA Trp aminoacylation.…”

“…When the acceptorstem of the tRNA binds to the catalytic center in one subunit, the anticodon loop simultaneously binds to the C-terminal domain in the other subunit (17)(18)(19)(20). The acceptor-stem binding domain is near the aminoacyl-AMP pocket (13,14). Therefore, the activated amino acid is carried conveniently to the 2Ј-OH of adenosine in the CCA terminus in tRNA.…”