Fidelity in Protein Synthesis

The effect of ribosome species on ambiguity during the transfer of amino acids from Escherichia coli aminoacyl transfer RNAs into protein has been examined under a variety of environmental conditions. Streptomycin greatly enhanced polyuridylic acid-directed transfer of leucine relative to phenylalanine (ambiguity ratio) with E. coli ribosomes, but had little effect with equivalent concentrations of reticulocyte ribosomes. The addition of E. coli supernatant fraction did not increase the ambiguity ratio obtained with reticulocyte ribosomes. In the presence of streptomycin, ambiguity was maximal at low concentrations of transfer RNA with E. coli ribosomes; transfer RNA concentration had a lesser effect on the low ambiguity obtained with reticulocyte ribosomes. Raising the magnesium concentration from 6 to 13 x 10-3 m increased the ambiguity ratio from 9 to 63% with E. coli ribosomes and from 0 to 25% with reticulocyte ribosomes. Ethanol increased the ambiguity ratio with E. coli ribosomes but not with reticulocyte ribosomes. The high fidelity of reticulocyte ribosomes was not restricted to leucine-phenylalanine ambiguity; although streptomycin caused a copolymer of uridylic and guanylic acid to miscode for arginine with E. coli ribosomes, this effect was not observed with reticulocyte ribosomes. These results show that streptomycin, a low concentration of transfer RNA, a high concentration of magnesium, and the addition of ethanol exert their miscoding effects at the ribosomal level. The fact that reticulocyte ribosomes are more resistant to these miscoding effects than E. coli ribosomes suggests that the ribosome plays an active role in codon recognition.