In vitro formation of beta-galactosidase with a template containing the lac genes fused to gene ilvD

Abstract

An in vitro coupled transcription-translation system was used to synthesize transaminase B and beta-galactosidase in the presence of a deoxyribonucleic acid template containing lac deoxyribonucleic acid under normal lac-specific control and in the presence of several deoxyribonucleic acid templates containing lac deoxyribonucleic acid fused to the ilvD gene. Time course experiments revealed that transcription of the lacZ gene from the fusion template required a longer time than did that initiated at the lac promoter. With a phage template containing an intact ilvE gene but lacking the normal ilv-specific promoter, synthesis of ilvE message was completed before synthesis of lacZ message. A phage template that contained the normal ilv-specific promoter but from which part of ilvE had been deleted also allowed formation of beta-galactosidase. Three plasmids containing the ilv-lac fusion were also used as templates. Two plasmids that contained both an intact ilvE gene and the normal ilv-specific promoter required longer times for lacZ transcription but were more efficient templates than was a plasmid in which the ilv-lac fusion, the ilvE gene, and the contiguous non-specific ilvE promoter were inverted with respect to the normal ilv-specific promoter. beta-Galactosidase synthesis was stimulated by guanosine 3'-pyrophosphate-5'-pyrophosphate with all templates tested except that in which the ilv-lac fusion had been inverted. Presumptive evidence was obtained for the generation of a limiting isoleucine signal by incorporating inhibitors of isoleucyl transfer ribonucleic acid synthetase into the coupled transcription-translation system.