Construction of a FRS1-FRS2 operon encoding the structural genes for the alpha and beta subunits of yeast phenylalanyl-tRNA synthetase and its use in deletion analysis.
FRS1 and FRS2, the structural genes encoding the large (alpha) and small (beta) subunits of yeast phenylalanyl-tRNA synthetase (PheRS) were placed under the control of the lacZ promoter by creating an artificial operon. The FRS2 gene was fused next to the promoter, followed by a 14 base pair intergenic sequence containing a translation reinitiation site in front of the FRS1 coding sequences. The engineered PheRS has 16 N-terminal amino acids from beta-galactosidase fused to the beta subunit. However, the purified protein shows a Km value for tRNA(Phe) that is indistinguishable from that of the the native enzyme. The product of the FRS2-FRS1 operon is not able to complement thermosensitive E. coli PheRS, indicating the lack of heterologous aminoacylation in vivo. We made a deletion in the FRS2 gene that removed about 150 amino terminal residues of the beta subunit. The truncated protein showed intact ATP-PPi exchange, whereas tRNA aminoacylation was lost. This result is similar to that of limited proteolysis performed on the native enzyme that yielded a tetrameric alpha 2 beta'2 structure, able to form aminoacyladenylate but unable to bind tRNA(Phe). A deletion of 50 amino acids from the carboxyl terminus of the beta chain resulted in the loss of both enzyme activities; this suggests the participation of the C-terminal end of the beta subunit in the active site or in subunit assembly to yield a tetrameric functional enzyme.
Sanni
A
A
Institut de Biologie Moléculaire et Cellulaire, Laboratoire de Biochimie, Strasbourg, France.
Walter
P
P
Ebel
J P
JP
Fasiolo
F
F
D000215:Acylation; D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics*; D001483:Base Sequence; D015153:Blotting, Western; D002872:Chromosome Deletion; D015825:Chromosomes, Fungal; D003001:Cloning, Molecular; D005800:Genes, Fungal; D007700:Kinetics; D008969:Molecular Sequence Data; D009876:Operon; D010652:Phenylalanine-tRNA Ligase / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D015003:Yeasts / Q000201:enzymology / Q000235:genetics*
D000604:Amino Acyl-tRNA Synthetases; D010652:Phenylalanine-tRNA Ligase