In vitro synthesis of cystathionine gamma-synthetase in Escherichia coli K-12

Abstract

Synthesis of cystathionine gamma-synthetase directed by DNA from a lambdadmet transducing phage has been achieved in cell extracts from Escherichia coli K-12. Enzyme synthesis was stimulated two- to threefold by the addition of guanosine 3'-diphosphate 5'-diphosphate to the incubation mixtures. Kinetic studies showed a 1.5- to 2.0-min lag between initiation of transcription and completion of a translatable message. This lag is shorter than that observed for beta-galactosidase synthesis with DNA from a lac transducing phage known to initiate transcription at the lac promoter. This result, together with information on the structure of the transducing phage, shows that pL is not used for initiation of in vitro metB transcription. Attempts to demonstrate repression were not successful, and unexpectedly, extracts from metJ+ strains were found to be more effective at enzyme synthesis than those from their metJ derivatives.