Abstract
can esterify serine to all of the purified isoacceptor RNAs. However, the extent of charging is only a small fraction of the level obtained with the homologous seryl-tRNA synthetase. It has been well established that most amino acids are speci- fied by more than one codon. A single tRNA molecule, how- ever, can recognize only one, two, or three codons. Thus, the proper translation of the genetic message to an amino acid specified by four codons, for example, requires at least two tRNA species (1, 2). This explains the occurrence in cells of families of tRNAs with different coding response but specifying the same amino acid (isoaccepting tRNAs). The number of such isoacceptors is further increased by the existence of multiple tRNA species specific for the same ammo acid and the same codon (redundant tRNAs (2)). Since leucine, serine, and arginine each have six codons, a larger number of isoacceptor RNAs is expected for these amino acids than for all others. Of these, the family of serine tRNAs has been most carefully studied, and there are three reports (3-5) of separation and partial puri- fication of the tRNASer family from Escherichia coli. This report describes a more extensive separation and purifi- * This work was supported by Grants GM 15401 and FR-07015 from the United States Public Health Service, National Institutes of Health, and Grant GB 7269 from the National Science Foun- dation. $ Centennial Fellow
Highlights
Our studies show that the heterologous aminoacyl-tRNA synthetases recognize all five E. co& tRNASer species, but that charging of them is not carried to completion, even in the presence of an excess of enzyme
A rapid purification of the mixture of all E. coli tRNASer species can be achieved by column chromatography on benzoylated DEAE-cellulose in the absence of Mg++ as previously described [17]
The salt gradient elutes the bulk of the tRNA which is almost devoid of tRNASer The alcohol wash removes from the column the remaining tRNA which includes all tRNASer The purification of the mixture of tRNASe’ is about 7-fold in this step
Summary
General-Uniformly labeled L-[14C]serine with a specific activity of 160 mCi per mmole was obtained commercially. The trinucleoside diphosphates used were prepared by chemical methods which have been described [9]. Polyethyleneglycol (Carbowax 6000) was purchased from Union Carbide. Benzoylated DEAE-cellulose was prepared by the met.hod of Gillam et al [10]. Protein concentration was determined by the method of
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