Using random mutagenesis and a genetic screening in
yeast, we isolated 26 mutations that inactivate Saccharomyces
cerevisiae arginyl–tRNA synthetase (ArgRS).
The mutations were identified and the kinetic parameters
of the corresponding proteins were tested after purification
of the expression products in Escherichia coli.
The effects were interpreted in the light of the crystal
structure of ArgRS. Eighteen functional residues were found
around the arginine-binding pocket and eight others in
the carboxy-terminal domain of the enzyme. Mutations of
these residues all act by strongly impairing the rates
of tRNA charging and arginine activation. Thus, ArgRS and
tRNAArg can be considered as a kind of ribonucleoprotein,
where the tRNA, before being charged, is acting as a cofactor
that activates the enzyme. Furthermore, by using different
tRNAArg isoacceptors and heterologous tRNAAsp,
we highlighted the crucial role of several residues of
the carboxy-terminal domain in tRNA recognition and discrimination.