Nucleic Acids Research, 1990, Vol. 18, No. 8 2087-2092
© 1990
ENZYMOLOGY |
Construction of a FRS1-FRS2 operon encoding the structural genes for the 
and ß subunits of yeast phenylalanyl-tRNA synthetase and its use in deletion analysis
Institut de Biologie Moléculaire et Cellulaire, Laboratoire de Biochimie 15 rue R.Descartes, 67084 Strasbourg Cedex, France
* To whom correpondence should be addressed
Received December 19, 1989. Revised March 20, 1990. Accepted March 20, 1990.
FRS1 and FRS2, the structural genes encoding the large (
) and small (ß) subunits of yeast phenylalanyltRNA 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 ß-galactosidase fused to the ß subunit. However, the purified protein shows a Km value for tRNAphe 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 ß 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 2ß'2 structure, able to form aminoacyladenylate but unable to bind tRNAPhe. A deletion of 50 amino acids from the carboxyl terminus of the ß chain resulted in the loss of both enzyme activities; this suggests the participation of the C-terminal end of the ß subunit in the active site or in subunit assembly to yield a tetrameric functional enzyme.