In vitro synthesis and and regulation of the biotin enzymes of Escherichia coli K-12.
The synthesis and regulation of two of the enzymes of the biotin operon of Escherichia coli, 7,8-diaminopelargonic acid aminotransferase and dethiobiotin synthetase, were studied in vitro in a coupled transcription-translation system. These enzymes are encoded by genes located on opposite strands of the divergently transcribed operon (A. Guha, Y. Saturen, and W. Szybalski, J. Mol. Biol. 56:53-62, 1971). The kinetics of synthesis of both the enzymes were determined and the efficiency of the system was 0.3 to 0.4% that of the in vivo rate of synthesis in derepressed cells. Guanosine 3'-diphosphate 5'-diphosphate at 0.2 mM concentration stimulated the synthesis of 7,8-diaminopelargonic acid aminotransferase two- to threefold but had no effect on dethiobiotin synthetase synthesis. Biotin, which was most effective as the corepressor in vivo, also functioned in vitro at physiological concentrations in conjunction with a crude repressor protein isolated from a lysogen carrying the bioR gene. However, the two strands showed differential repression. At a repressor concentration where 7,8-diaminopelargonic acid aminotransferase synthesis was completely repressed, the repression of dethiobiotin synthetase was only 20% and did not exceed 50% with increasing repressor concentrations. Although the exact reason for the partial repression remains to be resolved, our data clearly suggest that the biotin operon is regulated from two separate operators.
D001710:Biotin / Q000031:analogs & derivatives / Q000096:biosynthesis; D019731:Carbon-Nitrogen Ligases; D002474:Cell-Free System; D004794:Enzyme Repression; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D008025:Ligases / Q000096:biosynthesis*; D009876:Operon; D010453:Peptide Synthases / Q000235:genetics; D012436:S-Adenosylmethionine; D000637:Transaminases / Q000096:biosynthesis* / Q000235:genetics; D014158:Transcription, Genetic
D001710:Biotin; D012436:S-Adenosylmethionine; C020029:7,8-diaminopelargonic acid aminotransferase; D000637:Transaminases; D008025:Ligases; D019731:Carbon-Nitrogen Ligases; D010453:Peptide Synthases; C020024:dethiobiotin synthetase