Quantities of individual aminoacyl-tRNA families and their turnover in Escherichia coli

Abstract

The cellular content of all 20 aminoacyl-tRNA species was determined in small cultures of Escherichia coli by labeling cells with 3H-amino acids and extraction of 3H-amino acid-labeled nucleic acid by standard procedures. Of 3H-amino acid-labeled material, 25 to 90% was identified as 3H-aminoacyl-tRNA by the following criteria: sensitivity to base hydrolysis with expected kinetics; association of 3H counts released by base treatment of the 3H-amino acid-labeled nucleic acid with amino acid standards upon paper chromatography of the hydrolysate; and changes in the amount of 3H-amino acid-labeled nucleic acid recovered from cells as a function of time. Individual aminoacyl-tRNA content was determined with as few as 8 X 10(7) to 4 X 10(8) E. coli cells. Although the total number of aminoacyl-tRNA molecules per cell varied only by 10 to 20% among various strains of E. coli, some individual aminoacyl-tRNA families varied two- to threefold among strains. For a given amino acid, the number of aminoacyl-tRNA molecules per cell in E. coli strain K38 growing with a doubling time of 60 min varied from 730 (glutamyl-tRNA) to 7,910 (valyl-tRNA) with a mean value of 3,200. The total number of aminoacyl-tRNA molecules per cell (6.4 X 10(4)) in E. coli K38 was 5.5-fold higher than the number of ribosomes and was equal to 84% of the amount of elongation factor Tu molecules per cell. The ratio of aminoacyl-tRNA to synthetase for 10 amino acids varied from about 1 to 15 with a mean value of 4.7. The turnover of individual aminoacyl-tRNA families in E. coli cells was estimated to be in the range of 1.7 to 8.1 s-1 with a mean value of 3.7 s-1. An estimate of minimum in vivo molecular activity of aminoacyl-tRNA synthetases gives values of 2 to 48 s-1 for individual enzymes.