Receptor site for the 5'-phosphate of elongator tRNAs governs substrate selection by peptidyl-tRNA hydrolase.
Eubacterial peptidyl-tRNA hydrolase (PTH) recycles all N-blocked aminoacyl-tRNA molecules but initiator formyl-methionyl-tRNAfMet, the acceptor helix of which is characterized by a 1-72 mismatch. Positive selection by PTH of noninitiator tRNA molecules with a full 1-72 base pair is abolished, however, upon the removal of the 5'-phosphate. The tRNA 5'-phosphate plays therefore the role of a relay between the enzyme and the status of the 1-72 base pair. In this study, the receptor site for the 5'-phosphate of elongator peptidyl-tRNAs and the position at the surface of PTH of the 3'-end of complexed peptidyl-tRNA are identified by site-directed mutagenesis experiments. The former site comprehends two cationic side chains (K105 and R133) which are likely to clamp the phosphate. The second corresponds to a four asparagine cluster (N10, N21, N68, and N114). By using these two positional constraints, the acceptor arm of elongation factor Tu-bound Phe-tRNAPhe could be docked to PTH. Contacts involve the acceptor and TPsiC stems. By comparing the obtained 3D model to that of EF-Tu:Phe-tRNAPhe crystalline complex in which the 5'-phosphate of the ligand also lies between a K and an R side chain, we propose that, in both systems, the capacity of the 5'-phosphate of a tRNA to reach or not a receptor site is the main identity element governing generic selection of elongator tRNAs. On the other hand, while the 1-72 mismatch acts as an antideterminant for PTH or EF-Tu recognition, it behaves as a positive determinant for the formylation of initiator Met-tRNAfMet.
Fromant
M
M
Laboratoire de Biochimie, UMR 7654 CNRS, Ecole Polytechnique, Palaiseau, France.
Plateau
P
P
Schmitt
E
E
Mechulam
Y
Y
Blanquet
S
S
D001665:Binding Sites / Q000235:genetics; D002265:Carboxylic Ester Hydrolases / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D002412:Cations; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010441:Peptide Chain Elongation, Translational / Q000235:genetics; D010449:Peptide Mapping; D010652:Phenylalanine-tRNA Ligase / Q000235:genetics / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism*; D010766:Phosphorylation; D013379:Substrate Specificity / Q000235:genetics
D002412:Cations; D010710:Phosphates; D002265:Carboxylic Ester Hydrolases; C019674:aminoacyl-tRNA hydrolase; D010652:Phenylalanine-tRNA Ligase