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Mechanism of initiation and repression of in vitro transcription of the lac operon of Escherichia coli.

A cyclic AMP-binding protein (CAP protein), cyclic AMP, and RNA polymerase holoenzyme are shown to initiate lac transcription at the lac promoter. Lac repressor appears to control transcription by preventing RNA polymerase and/or CAP protein from binding to the lac promoter. Results support the idea that the lac promoter is composed of two sites that interact with CAP protein and RNA polymerase holoenzyme. The promoter can be altered by mutation so that holoenzyme alone can initiate lac transcription correctly.

Cyclic AMP↗

Insertion of the tetracycline resistance translocation unit Tn10 in the lac operon of Escherichia coli K12.

The majority of TN10 insertions in the lacZ gene of Escherichia coli occurred in a small region of the promoter distal part of the gene. The resulting mutations were polar on lacY and reverted to Lac+ at a frequency of 10(-8). None of the revertants were Tcr. Furthermore Lac+ Tcr revertants could not be selected directly. Relief of polarity revertants of the lacZ::Tn10 mutants were formed at a frequency of 10(-5) - 10(-4). Most resulted from a deletion event internal to the transposon which removed the Tcr genes and the putative transcription terminator. It is postulated that a fragment of Tn10 remains at the original insertion point to cause a revertible Lac- mutation.

Chromosome Mapping↗

Regulation of lac operon expression: reappraisal of the theory of catabolite repression.

The physiological state of Escherichia coli with respect to (permanent) catabolite repression was assessed by measuring the steady-state level of beta-galactosidase in induced or in constitutive cells under a variety of growth conditions. Four results were obtained. (i) Catabolite repression had a major effect on fully induced or constitutive expression of the lac gene, and the magnitude of this effect was found to be dependent on the promoter structure; cells with a wild-type lac promoter showed an 18-fold variation in lac expression, and cells with the lacP37 (formerly lac-L37) promoter exhibited several hundred-fold variation. (ii) Exogenous adenosine cyclic 3',5'-monophosphoric acid (cAMP) could not abolish catabolite repression, even though several controls demonstrated that cAMP was entering the cells in significant amounts. (Rapid intracellular degradation of cAMP could not be ruled out.) (iii) Neither the growth rate nor the presence of biosynthetic products altered the degree of catabolite repression; all variation could be related to the catabolites present in the growth medium. (iv) Slowing by imposing an amino acid restriction decreased the differential rate of beta-galactosidase synthesis from the wild-type lac promoter when bacteria were cultured in either the absence or presence of cAMP; this decreased lac expression also occurred when the bacteria harbored the catabolite-insensitive lacP5 (formerly lacUV5) promoter mutation. These findings support the idea that (permanent) catabolite repression is set by the catabolites in the growth medium and may not be related to an imbalance between catabolism and anabolism.

Culture Media↗