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.

            

1. 

Gene Regulation Strategy In Bacteriophage:

• Bacteriophage lambda (λ) is a temperate phage that infects the bacterium E.coli. After infecting host cells, lambda phage can follow one of two alternative cycles: lytic and lysogenic cycles.
• Lysogeny maintaintains long term relationship between host bacterium and parasitic bacteriophage, hence, Bacteriophage have evolved gene regulation strategy to offer tight regulation of lytic pathway gene.
• Lambda bacteriophage have evolved tight regulation of lysogeny. They takes lysogenic induction (termination of lysogeny to lytic cycle) only when there are damages in bacterial DNA because of mutagens (mainly caused by UV radiation).
• Bacteria have evolved Restriction Endonuclease to impart defense against bacteriophage to counter this defense, many bacteriophages have evolved restriction site methylation.

GENES OF LAMBDA PHAGE:

• There are three classes of genes in phage genome that regulates whether the lytic or lysogenic cycle will emerge- 

1. Immediate early gene - Cro, CII & N gene
2. Delayed early genes - replication gene O, P and Q
3. Late genes

• Genes that favour the lysogenic and lytic cycle are:

1. Genes favouring lysogenic Cycle - cI, cII, cIII, int and N gene.
2. Genes favouring lytic Cycle - cro, Q and xis.

GENE FAVOURING LYSOGENY:

1. cI gene : 'c' stands for 'clear' as in clear plaque mutant. It was reported that bacterial colonies in culture were cleary developed when bacteriophage have wild type cI gene.

• cI mutation formed lytic plaque in colony.
• cI codes for "λ repressor" protein.
• λ repressor acts as strong repressor and offers tight regulation to cro gene.
• λ repressor made up of 236 amino acid residues and folds into a structure ressembling a dumbbell, with two domains separated.The C-terminal domains associate to form dimers, the N-terminal domains bind DNA. The repressor exists primarily as a homodimer.

Fig: A repressor binds to three sites in the right operator

• λ repressor can bind at three operators and sequence of binding affinity is OR1 > OR2 > OR3.
• λ repressor has strong affinity to bind at OR1 which is the operator of cro gene. Large number of λ repressor binds at OR1 due to the feature of dimerization & tetramerization thus tight blocking of cro gene and leaving no chance of leaky expression.
• λ repressor also acts as autoactivator as it binds at OR2 with medium affinity and activates α- NTD (N- terminal domain) of RNA polymerase of PRM (Promoter Repressor Maintenance) thus, increases its own production.
• λ repressor also acts as autorepressor as it can bind with poor affinity at OR3, thus, blocking transcription of cI but it happens only when the concentration of λ repressor is lysogenized bacterial cell increases upto 20 fold over threshold.
• PRM is the weak promoter, hence, it requires activation. PRM is activated by cII.

Fig: In the lysogenic state, Repressor are bound to OR1 and OR2, but not to OR3 which allows RNA polymerase to binds to PRM and trancribe the cI gene

2. cII gene : Transcribed by PL promoter (Promoter Leftward), cII protein acts as activator of PRM and it prepares PRM for RNA polymerase binding but cII is highly unstable protein because bacteria 'Hfl' protein (High frequency of lysogeny) causes degradation of cII.

• Hfl is an evolution of bacterial cell to prevent lysogeny.
• cIII protein is evolved by bacteriophage to protects cII from Hfl mediated degradation.

3. cIII gene : Transcribed by RNA polymerase of PRE (Promoter Repressor Establishment) 

• cIII protects cII from Hfl mediated degradation.

4. N-gene : Transcribed with cII & forms N-protein. 

• N- protein prevents Rho dependent transcription termination of cI gene. Thus, N-protein is an antiterminator of cI.
• N-protein binds at Rho utilisation sites of cI mRNA thus, blocking the binding of Rho and no premature transcription termination.

EXPRESSION SEQUENCE OF LYSOGENIC GENES:

               cIII → cII → N→ cI

LYSOGENIC INDUCTION:

• Termination of Lysogeny to lytic cycle.
• Lambda bacteriophage shows lysogenic induction when the cytosolic level of Rec A protein is increased in bacterial cell.
• Rec A protein is synthesized by bacterium when the amount of single stranded DNA increases.
• Single stranded DNA increased in bacterial cell due to-

1. Paused DNA polymerases at pyrimidine dimers while helicase unwinding the DNA.
2. Pick up of DNA through transformation or conjugation mechanism.
3. Increase in temperature may causes generation of ssDNA.

• λ repressor of bacteriophage has an evolutionary novality as it takes autocleavage upon interaction with Rec A.

Fig: Activated RecA protein cleaves repressor which then cannot dimerize

GENES FAVOURING LYTIC CYCLE:

• Upon autocleavage of λ repressor, cro gene is switched on.
• cro (Control of Repressor) gene acts as negative regulator of cIII, cII & cI. Futher, cro gene induces the expression of xis gene &Q gene.
• xis gene codes for excisase enzyme which cuts to separate the phage DNA from bacterial DNA.
• Q gene coded Q-protein acts as antiterminator of cro gene.

The decision between lysis and lysogeny is, therefore, determined by the outcome of a race between cI and cro. If the cI repressor is synthesized more quickly than the cro repressor, the lysogeny follows. However, if cro wins the race, then the cro repressor block cI expression before enough cI repressor has been synthesized to silence the cro gene. As a result, the phage follows the lytic cycle. The decision appear to the random, depending on chance events that lead to either the cI or the cro repressor accumulating the quickest in the cell.