LAC OPERON IN E.coli :

INTRODUCTION:

• Lac Operon  is an operon required for the transport and metabolism of lactose in E. coli.
• Lactose is a dissacharide made up of glucose and galactose.

• Lactose is a β- galactoside that E. coli can use for energy and as a carbon source after it is broken down into glucose and galactose.

• Lac operon is an inducible system,  

• Lac operon consists of an operator, a promoter and three structural genes.

• The lac operator is a short region of DNA that interacts with a regulatory protein lac repressor, which negatively controls the transcription of the operon.

• It is a cis-acting sequence on the DNA to which the lac repressor binds.

• When the lac repressor binds  the operator, it prevents RNA polymerase from initiating transcription at the promoter.

• A structural gene is any gene that codes for a protein (or RNA). Lactose uptake and degradation are mediated by the products of three structural genes. The roles of the three structural genes are:     

Lac Z :     It codes for the enzymes β - galactosidase. This enzyme has two function:

   (a). It catalyzes the breakdown of lactose into glucose and galactose,

   (b).  It catalyzes the conversion of lactose to a related form called allolactose.

  2.  Lac Y:

• It codes for the β- galactosidase permease.
•  Lactose permease (also called M- protein) is found in the E.coli membrane and is needed for the active transport of lactose from the growth medium into the cell.

  3.   Lac A :

• It codes for the β- galactosidase acetyltransferase (or β- galactosidase transacetylase). 
• Transacetylase is a protein whose function is poorly understood.

      Figure : The lac operon includes a promoter (P), an operator (O) and three structural genes- the lac Z, lacY and lacA. It is transcribed as multigenic (polycistronic) mRNA.

• Regulation of the transcription of this mRNA controls the synthesis of all three enzymes. This operon is controlled by both positive and negative regulation.

1. Negative Regulation of lac operon :

• In absence of lactose (termed inducer), the lac repressor (product of lac I gene) binds to the lac operator and prevents transcription initiation of the lac operon. Because binding of an active repressor prevents the transcription, it is termed negative regulation.

• The lac repressor is the product of trans- acting regulatory gene (lacI) that regulates the transcription of the structural genes comprising lac operon.

• Isopropylthiogalactoside (IPTG), a synthetic non- metabolizable analog of the lactose, also acts as an inducer.

1. In absence of lactose, the lac repressor binds operator and represses transcription from the lac operon.
2. In presence of lactose, few molecules of enzyme β- galactosidase present in the cell convert lactose into allolactose. Allolactose binds to the lac repressor, leading to inactivation of operator binding site and to production of lac mRNA.

Figure : Induction of the lac operon 

     2.   Positive Regulation And Catabolite Repression:

• When glucose is available as an energy source, it is used in preference to other sugars. So, when E. coli finds both glucose and lactose in the medium, it metabolizes the glucose and represses the use of lactose. 

• In the presence of both lactose and glucose, the synthesis of β- galactosidase is not induced until all the glucose has been used up. 

• The ability of glucose to inhibit the synthesis of certain enzymes, referred to as the glucose affect. This phenomenon is due to the repressive effect of glucose on the synthesis of enzymes required for the metabolism of other sugars. Later, the term catabolite repression was introduced as a general name for the glucose effect.

• Catabolite repression works through an activator protein. Catabolites repression brought about the discovery of a positive regulation of transcription by the cAMP receptor protein, CRP (also called the catabolite gene activator protein, CAP).

•  CRP is a homodimer and acts as an activator. It is activated by binding of a single molecule of cAMP. It is needed for RNA polymerase to initiate transcription of many operons of E.coli..

• cAMP is synthesized by the enzyme adenylate cyclase, and its concentration is related to glucose concentration. So, when the glucose level in the cell is high, the cAMP concentration is low.

• Conversely, when the glucose level is low, the cAMP concentration is high. By itself, CAP cannot bind to the CAP binding site of the lac operon. 

• However, by binding to its allosteric effector, cAMP, CAP is able to bind to the CAP binding site and activate transcription.            

       

Figure : Catabolite repression of lac operon.

•  Glucose concentration regulate cAMP concentration,

High glucose concentration → Low cAMP concentration → Inactive CAP

Low glucose concentration → High cAMP concentration → Active CAP            

Figure : Expression of the lac operon. The presence or absence of the sugars lactose and glucose control the level of expression of the lac operon. Above basal levels of expression require the presence of lactose and absence of the preferred energy source, glucose.

            

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