Regulation of formation of threonyl-tRNA synthetase, phenylalanyl-tRNA synthetase and protein synthesis initiation factor 3 from Escherichia coli in vivo and in vitro

Abstract

The expression of the structural genes for the protein synthesis initiation factor 3 (IF-3), threonyl-tRNA synthetase and phenylalanyl-tRNA synthetase carried by the transducing phage lambda p2 was studied in a DNA-dependent transcription-translation system in vitro and the results were compared to the regulatory pattern in vivo. In vitro, the DNA of the phage lambda p2 gives rise to the formation of the two forms of IF-3 (IF-31 and IF-3S) which are known to be present in vivo. The kinetics of synthesis indicate an interconversion of IF-31 into IF-3S. Addition of excess purified IF-31 does not significantly repress IF-3 synthesis but does stimulate the rate of conversion of IF-31 into IF-3S. This apparent lack of autoregulation in vitro is in accordance with gene-dosage-dependent synthesis in vivo. The fact that strains with more than one copy of the IF-3 structural gene contain a higher relative amount of IF-3S than do haploid ones suggests that the proteolytic conversion of IF-31 into IF-3S may occur predominantly in the free (non-ribosome-bound) state. In vivo, the amount of IF-3 varies with the growth rate much like elongation factor Tu or aminoacyl-tRNA synthetases. As with the aminoacyl-tRNA synthetases, IF-3 synthesis is not significantly subject to a stringent control system. This coordinated regulatory response in vivo, however, is not paralleled by the susceptibility of synthesis in vitro to guanosine 3'-diphosphate 5'-diphosphate (ppGpp), since IF-3 formation is inhibited by ppGpp whereas that of threonyl-tRNA synthetase and phenylalanyl-tRNA synthetase is stimulated.