The Lac Operon

The regulatory mechanism of operon responsible for utilization of lactose as a carbon source is called the Lac Operon. It was extensively studied for the first time by Jacob and Monod (1961). Commonly an operon consists of repressor, promoter, operator and structural genes. The message produced by an operon is polycistronic because the information of all the structural genes resides on a single molecule of m RNA. The lactose utilizing system consists of two types of components: the structural genes (LacZ, LacY and LacA) the products of which are required for transport and metabolism of lactose and the regulatory genes (the lacI, the lacO and the lacP). These two components together comprises the lac operon. The Structural Genes: The number of structural genes corresponds to the number of proteins. Lac operon consist of three structural genes i.e. lac Z, lac Y and lac A. Lac lac z codes for the enzyme beta galactosidase (β-gal), which breaks down lactose into glucose and galactose and, also converts lactose to allolactose. This enzyme is a tetramer of four identical subunits each with molecular weight of 1,16,400. lac Y codes for permease, which is an enzyme needed for transporting lactose into the cell. Permease has molecular weight of 46,500. It is hydrophobic. lac A codes for transacetylase, which is not involved in lactose metabolism. The molecular weight of enzyme transacetylase is 30,000.

The Regulatory Genes: It consist of lacI, lacP and lacO. O is the operator region, which, has the ability to bind to a repressor protein. P is the promoter, which overlaps with the operator. If the repressor protein is bound to the operator, transcription does not occur because the repressor blocks the promoter region , so that RNA pol cannot bind to initiate transcription. Associated to the lac operon is the lac i gene which is located upstream from the promoter/operator, and codes for the repressor protein that binds to the operator. This protein is a tetramer (it has 4 sub-units). The repressor is an allosteric protein, which means that it changes its shape in response to binding of an inducer molecule. In this case the inducer is allolactose, a metabolite produced by conversion of lactose by b-gal. When the inducer binds to the repressor, it changes in shape and no longer can bind to the operator. Then RNA pol can bind to promoter to initiate transcription. the

Repressed state of lac operon: In repressed state, the repressor protein bind to the operator and form an operator-repressor complex which in turn physically blocks the transcription of Z, Y and A genes by preventing the release of RNA polymerase to begin transcription.

Induced state of lac operon: When an inducer (eg. Lactose) is present, it binds to repressor proteins and forms an inducerrepressor complex. This complex cannot bind to the operator. Due to the formation of complex the repressor undergoes

changes in conformation of shape and becomes inactive. Consequently, polycistronic (proteins). the mRNAs structural and the genes later can synthesize the



Positive Regulation of the Lac Operon-Catabolic Control:
Cyclic AMP (c AMP) is the small molecule which is distributed in animal tissues, and controls the action of many hormones. It is also present in E.coli and the other bacteria. The c AMP is synthesized by the enzyme adenyl cyclase. Its concentration is directly regulated by glucose metabolism. cAMP is required to activate an allosteric protein called CAP (catabolite activator protein) which binds to the promoter CAP site and stimulates the binding of RNAp polymerase

to the promoter for the initiation of transcription. Thus, to efficiently promote gene transcription of the lac operon, not only must lactose be present to inactivate the lac repressor, but cAMP must be available to bind to CAP

which binds to DNA to facilitate transcription. In the presence of glucose, adenylate cyclase (AC) activity is blocked. AC is required to synthesize cAMP from ATP. Therefore, if cAMP levels are low, CAP is inactive and transcription does not occur. In the absence of glucose, cAMP levels are high, CAP is activated by cAMP, and transcription occurs (in the presence of lactose). The promoter region consists of palindromic sequence of

nucleotides that are recognized by a protein called CRP (cyclic AMP receptor protein). It has been shown that CRP binds to cAMP molecule and form a cAMP-CRP complex. This complex binds to the promoter and enhances the attachment of RNA polymerase to the promoter. Therefore, it increases transcription and translation processes. Thus cAMP-CRP is a positive regulator in contrast with the repressor and the lac operon is controlled both positively and negatively.

Catabolite repression is positive control of the lac operon. The effect is an increase in the rate of transcription. In this case, the CAP protein is activated by cAMP to bind to the lac operon and facilitate the binding of RNA polymerase to the promoter to transcribe the genes for lactose utilization.

The activity of the lac operon is also affected by the presence or absence of glucose in the media. If both, glucose and lactose are present in the media, bacteria will prefer to use glucose over lactose. Glucose provides a positive control system superimposed to the lactose repressor-operator system. If both glucose and lactose are present, synthesis of b-gal will not be induced until all the glucose in the media is depleted. This explains why the lac operon is off in spite of being under negative control. When glucose levels are high, levels of the compound cAMP (cyclic adenosine monophosphate) are low and vice versa. High levels of cAMP, which occur when glucose is low, are necessary for activation of the lac operon. cAMP is a glucose catabolite (compound resulting from glucose breakdown during its utilization) derived from ATP, and capable of binding to a catabolite activation protein (CAP) produced by a gene called crp. CAP forms a complex with cAMP, which activates the lac operon by binding to a site in the promoter p called CAP site. When glucose is present, cAMP is low and cannot effectively bind to CAP. When CAP-cAMP is not bound to the promoter, transcription will not occur in the lac operon. Thus, bacteria will not produce b gal in the presence of glucose, whether lactose is present or not.

If glucose is low, the opposite is true. cAMP will be high, binding to CAP and allowing transcription of the lac operon. Therefore, cAMP is an activator of the lac operon, which turns it on. The following table shows the status of operator and CAP regions in varying glucose and lactose conditions:

Glucose Lactose Yes No Yes No No No Yes Yes

Promoter CAP bound CAP bound

Operator Repressor bound

Lac Transcription No No

CAP not bound Repressor bound

CAP not bound Repressor not bound No Repressor not bound Yes

Tryptophan operon
The genes for tryptophan biosynthesis in Escherichia coli are organized on the bacterial chromosome in the tryptophan operon (trp operon). An operon is a cluster of genes that are controlled by the same elements and which are coordinately transcribed and translated. The trp operon consists of a Promoter (P) region, an Operator (O) region, an Attenuator (A) region, and the five structural genes for the enzymes involved in tryptophan biosynthesis (Trp A-E).

Genetic organization of the Trp operon and its control elements.

R = Regulatory gene that encodes for the trp Repressor protein that is concerned with regulating the synthesis of the 5 gene products. An active

repressor binds to a specific nucleotide sequence in the operator region and thereby blocks binding of RNAp to the promoter to initiate transcription. O = Operator specific nucleotide sequence on DNA to which an active Repressor binds. P = Promoter specific nucleotide sequence on DNA to which RNA polymerase binds to initiate transcription. If the repressor protein binds to the operator, RNAp is prevented from binding with the promoter and initiating transcription. Therefore, none of the enzymes concerned with tryptophan biosynthesis are synthesized. A = Attenuator DNA sequence which lies between the operator and the structural genes for trp biosynthesis. The attenuator is a barrier that RNA polymerase must traverse if it is to transcribe the genes for tryptophan biosynthesis. In the presence of trp, most RNAp molecules fall off the DNA before transcribing the trp genes. In the absence of trp, RNAp is able to traverse the attenuator region to successfully transcribe the trp genes. Trp A, B, C, D, E = Structural genes for enzymes involved in tryptophan biosynthesis. Trp = tryptophan end product of the biosynthetic pathway. When combined with the repressor protein the Repressor is active. Trp is called a corepressor.

The trp operon is regulated by a regulatory gene (Trp L) associated with the trp promoter. The product of the Trp L gene is the trp Repressor, an allosteric protein which is regulated by tryptophan. The Repressor is produced constitutively in small amounts in an inactive form. When the Repressor combines with tryptophan it becomes activated and binds to the DNA of the trp operon in such a way that it blocks the transcription of the structural genes for tryptophan. Thus, in the presence of tryptophan, transcription of the genes for tryptophan biosynthesis are repressed (tryptophan is not produced), while in the absence of tryptophan, the genes for tryptophan biosynthesis can be transcribed (tryptophan is produced); See Figure 6 below.

Derepression of the trp operon. In the absence of trp the inactive repressor cannot bind to the operator to block transcription. The cell must synthesize the amino acid.

Repression of the trp operon. In the presence of tryptophan the trp operon is repressed because trp activates the repressor. Transcription is blocked because the active repressor binds to the DNA and prevents binding of RNA polymerase.

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