Yeast Phenylalanyl‐tRNA Synthetase

Abstract

The localization of the binding sites of the different ligands on the constitutive subunits of yeast phenylalanyl-tRNA synthetase was undertaken using a large variety of affinity and photoaffinity labelling techniques. The tRNAPh' was cross-linked to the enzyme by non-specific ultraviolet irradiation at 248 nm, specific irradiation in the wye base absorption band (315 nm), irradiation at 335 nm, in the absorption band of 4-thiouridine (s"U) residues introduced in the tRNA molecule, or by Schiffs base formation between periodate-oxidized tRNAPh' (tRNA;:') and the protein. ATP was specifically incorporated in its binding site upon photosensitized irradiation.The amino acid could be linked to the enzyme upon ultraviolet irradiation, either in the free state, engaged in the adenylate or bound to the tRNA.The tRNA, the ATP molecule and the amino acid linked to the tRNA were found to interact exclusively with the 0 subunit ( M , 63000). The phenylalanine residue, either free or joined to the adenylate, could be cross-linked with equal efficiency to either type of subunit, suggesting that the amino acid binding site is located in a contact area between the two subunits. The Schiffs base formation between tRNA:,he and the enzyme shows the existence of a lysyl group close to the binding site for the 3'-terminal adenosine of tRNA. This result was confirmed by the study of the inhibition of yeast phenylalanyl-tRNA synthetase with pyridoxal phosphate and the 2',3'-dialdehyde derivative of ATP, oATP.

Mechanism of translocation: relative arrangement of tRNA and mRNA on the ribosome.

“…The experiment exploits the photoreactivity of wybutine (Y-Wye), which occurs in tRNAPhe from yeast at the 3' side of the anticodon (10). The results demonstrate that the orientation of Y-Wye with respect to the mRNA is the same, or at least very similar, before and after translocation.…”

Arginyl‐Transfer Ribonucleic Acid Synthetase of Bacillus stearothermophilus. Purification and Kinetic Analysis

“…Mulvey and Fersht [22] and Baltzinger et al [23] have shown that certain monomeric and tetrameric enzymes act functionally as dimers. Thus, it is possible that the monomeric arginyl-tRNA synthetase is functional as a tRNA"rgdependent dimer.…”

Identification of the major tRNAPhe binding domain in the tetrameric structure of cytoplasmic phenylalanyl‐tRNA synthetase from baker's yeast

“…Secondly, earlier binding studies showed that UV irradiation of various (enzyme, tRNAPhe) complexes always led to an exclusive covalent labelling of the fi subunit: for instance s4U-tRNAPhe could be joined top and this suggests the existence of several contact areas between the protein and the ligand since s4U-tRNAPhe contained 5 to 6 modified uridines statistically distributed along the nucleotide sequence; apparently the interaction between ,& and this modified tRNA did not involve the 3 ' end and the catalytic site because no loss of ATP-PPi exchange activity was observed after photoirradiation of the complex [12].…”

“…This sequence is part of the fl' domain of p and it must be located near or in the active site. Indeed labelling this lysine resulted in a complete loss of the aminoacylation activity whilst the ATP-PPi exchange reaction remained unaffected [12]. It is worth stressing that this lysine 325 of p is located between two regions of high homology with the small subunit of phenylalanyl-tRNA synthetase from E. coli [ 141.…”

“…It is difficult to extend this model to the yeast phenylalanyl-tRNA synthetase because its isolated (Y or ,8 subunits are unable to catalyse the ATP-PPi exchange and tRNAPhe aminoacylation [ 111. Its active site is more likely built by the subunits interface as suggested by the localization of phenylalanine binding sites [12]: indeed phenylalanine, either free or as phenylalanyladenylate, could be cross-linked with an equal efficiency to either type of subunit.…”