Amplification of maize ribulose bisphosphate carboxylase large subunit synthesis in E. coli by transcriptional fusion with the lambda N operon.
The maize chloroplast gene coding for the large subunit of ribulose bisphosphate carboxylase (3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39) has been placed under the transcriptional control of the bacteriophage lambda promoter PL, by fusion with the lambda N operon located on a multicopy plasmid. Transcription from PL was repressed at 32 degrees C by the presence in the E. coli chromosome of a cIts gene that specifies a temperature-sensitive repressor. After inactivation of the repressor at 45 degrees C unmoderated transcription of the chloroplast gene occurred from the PL promoter. Translation was probably initiated from a chloroplast Shine-Dalgarno sequence located five nucleotides from the N-terminal methionine initiation codon to yield a polypeptide the same size as that synthesised in maize. This direct translation results in a level of expression of the chloroplast gene corresponding to approximately 2% of the total E. coli cell protein as ribulose bisphosphate carboxylase large subunits. Transcriptional fusions with the lambda N operon should provide a generally applicable, simple method for the amplification and regulation of chloroplast gene expression in E. coli.
D010582:Bacteriophage lambda / Q000235:genetics*; D002262:Carboxy-Lyases / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D005784:Gene Amplification; D007700:Kinetics; D009876:Operon; D010944:Plants / Q000201:enzymology* / Q000235:genetics; D010957:Plasmids; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics*; D014158:Transcription, Genetic; D003313:Zea mays / Q000201:enzymology / Q000235:genetics
D002262:Carboxy-Lyases; D012273:Ribulose-Bisphosphate Carboxylase