Lactose operon promoter

Van Rooijen, R. J., Gasson, M. J., De Vos, W. M. (1992). Characterization of the Lactococcus lactis lactose operon promoter contribution of flanking sequences and LacR repressor to promoter activity. Journal of Bacteriology, 174, 2273-2280. 

Fig. 24.5 Insertion of a cloned insulin gene into a vector carrying a bacterial promoter. The arrow indicates the direction of transcription. If we suppose the bacterial promoter is derived from the lactose operon then transcription will be initiated only in the presence of lactose.

A variety of different promoters have been used for the expression of antibody genes. Widely used is the lacZ promoter (lacZ) derived from the lactose operon (53). The gill promoter (gill) from the bacteriophage M13 (9), the tetracycline promoter (IX teto/p ref. 54) and the phoA promotor of the E. coli alkaline phosphatase (47) also have been used successfully. It appears that very strong promoters, for example, the synthetic promoter PAI/04/03 (55), are... 

The regulation of bacterial transcription is well illustrated by the lactose operon (lac operon) of the colon bacterium Escherichia coli in which the upstream region successively (from the 5 end of the sense strand) includes a promoter (P ) for the gene (I) coding for a repressor protein (the lac repressor), a CRP binding site , the promoter for the lac operon (P), and finally an operator site (O) that prefaces the Z, Y and A structural genes of the operon ... 

Figure 31.3. Operons. (A) The general structure of an operon as conceived by Jacob and Monod. (B) The structure of the lactose operon. In addition to the promoter (p) in the operon, a second promoter is present in front of the regulator gene (/) to drive the synthesis of the regulator.

See also The Genetic Code, Structure of tRNAs, Initiation of Translation, Prokaryotic Translation Regulation, Lactose Operon Regulation (from Chapter 26), Promoter Organization... 

CAP is a positive control element. When the level of glucose in cells is low, the level of cAMP is high, leading to the formation of cAMP-CAP complex. The cAMP-CAP complex binds to DNA in the promoter region, creating an entry site for RNA polymerase. The result is the transcription of the lactose operon (providing that no repressor is present) (see Figure 31.10 on p. 873 of the text). Coactivators act as positive control elements in eukaryotic transcription (see pp. 881-882 in the text). 

Fig. 9-17 The lactose operon of E. coll. Here I, p, o, z, y, and a denote the repressor gene, promoter, operator, p-galactosidase gene, permease gene, and transacetylase genes, respectively. Because the three genes, z, y and a, are transcribed as a single unit polycistronic mRNA), they are said to be expressed coordinately. When transcription is blocked by the binding of the repressor to the operator, none of the genes are expressed.

Enhanced gene expression can be achieved by the addition of a promoter to the cloned gene. For example, the lac promoter may be added upstream of the protein DNA sequence, giving rise to the expression of the protein in the presence of lactose and in the absence of glucose, (cf. operon hypothesis Section S.7). 

Catabolite activator protein, CAP (also called cAMP receptor protein, CRP) is an activator required for high level transcription of the lac operon. The active molecule is a CRP dimer that binds 3 5 cyclic AMP to form a CRP-cAMP complex. CRP-cAMP binds to the lac promoter and increases the binding of RNA polymerase, stimulating transcription of the lac operon. CRP dimer without cAMP cannot bind to this DNA. The action of CRP depends upon the carbon source available to the bacterium. When glucose is present, the intracellular level of cAMP falls, CRP cannot bind to the lac promoter and the lac operon is only weakly transcribed. When glucose is absent, the level of intracellular cAMP rises, the CRP-cAMP complex stimulates transcription of the lac operon and allows lactose to be used as an alternative carbon source. 

Figure 13.1 Operon model for control of protein synthesis. The example chosen is the lactose (lac) operon. I, regulatory gene p, promoter site o, operator gene z, y, and a represent the structural genes for j8-galactosidase, permease, and transacetylase, respectively.

There is also an aspect of positive control in the lac operon. The catabolite activator protein (CAP), carrying bound cAMP, is required for the binding of RNA polymerase to the promoter i.e., it has a direct, positive effect on transcription. However, relief of repression (i.e., induction) will not occur in the presence of glucose, because glucose lowers the level of cAMP, so that CAP is unable to exert its effect. This reflects the preference of the cell to use glucose rather than lactose as a carbon source. Thus it can be seen that the cell stringently controls expression of the lac genes it expresses them only if it needs to metabolize lactose. 

B. cAMP is involved in catabolite repression. Bacterial cells preferentially use glucose. When glucose is low, cAMP rises. cAMP binds to a protein and complexes near the lac promoter region, facilitating binding of RNA polymerase. Lactose must be present to inactivate the repressor, so that the operon may be expressed. 

B. In the absence of glucose and the presence of lactose, the lac repressor will be inactive, cAMP levels will rise, and CAP protein will bind to the lac promoter stimulating transcription of the operon. Tryptophan levels in the cell will be low, so the repressor for the trp operon will be inactive and the operon will be transcribed by RNA polymerase. Attenuation of transcription of this operon will decrease. 

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