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Class Notes

Introduction

• E. coli is a bacteria that lives in your colon. It has a metabolic pathway that allows for the synthesis of the amino acid tryptophan (Trp).
  • This pathway starts with a precurser molecule and proceeds through five enzyme catalyzed steps before reaching the final Trp product.
• It is important that E. coli be able to control the rate of Trp synthesis because the amount of Trp available from the environment varies considerably.
  • If you eat a meal with little or no Trp, the E. coli in your gut must compensate by making more.
  • If you eat a meal rich in Trp, E. coli doesn't want to waste valuable resources or energy to produce the amino acid because it is readily available for use.
• Therefore, E. coli uses the amount of Trp present to regulate the pathway.
  • If levels are not adequate, the rate of Trp synthesis is increased.
  • If levels are adequate, the rate of Trp synthesis is inhibited.
• There are two ways of regulating the Trp pathway:
  • The first method works to decrease the synthesis of Trp by inhibiting the first enzyme in the pathway, preventing the rest of the pathway from proceeding.
  • What inhibits the first enzyme? Trp does!
    • The more Trp in the cell, the more that can bind to the first enzyme and prevent it from catalyzing the first step.
  • This method of regulation is feedback inhibition in which the end product of a pathway acts as an inhibitor of an enzyme in that pathway.
• The other method of control stops the production of the enzymes in the pathway at the transcription level.
  • Remember that enzymes are proteins that must be transcribed and translated from the genetic code.
  • If the genes for the enzymes are not transcribed to mRNA, then translation to the enzymes cannot occur.
  • Without enzymes, there is no Trp synthesis.
• This method of control is called regulation of gene expression because control is taking place at the genetic level. This method of control will now be examined in detail.

Trp Operon

Repressible vs Inducible Systems

• The Trp pathway is anabolic as Trp is being synthesized. The Trp and other regulated anabolic pathways are usually repressible because the system can be repressed by an overabundance of the end product.
  • The end product, Trp, in this case, decreases or stops the transcription of the enzymes necessary for its production.
• Regulated catabolic pathways, on the other hand, are usually inducible because the pathway is stimulated rather than inhibited by a specific molecule. An example of an inducible system is lactose metabolism.

The Lac Operon

• The genes that code for the enzymes needed for lactose catabolism are clustered on the same chromosome in what is called the Lac operon.
• The Lac operon has three components:
  • Three Structural Genes:
    • These contain the genetic code for the three enzymes in the lac catabolic pathway
  • One Promoter:
    • DNA segment where RNA polymerase binds and starts transcription
  • One Operator:
    • DNA segment found between the promoter and structural genes.
    • It determines if transcription will take place.
    • If the operator in turned "on", transcription will occur.
• As in the Trp operon, the Lac operon is turned "off" by a specific protein called the repressor.
  • The repressor is the product of the regulator gene which is found outside the operon.
    • Transcription of the regulator produces mRNA which is translated into the repressor.
  • But unlike the Trp operon, the repressor is active in this form and does not require a co-repressor.
  • The active repressor binds to the operator blocking the advancement of RNA polymerase to the structural genes.
  • Without RNA polymerase, transcription and translation of the genes can't occur and the enzymes needed for Lac metabolism are not made.
• What turns the Lac operon "on"? Lactose does!
  • This makes sense because the cell only needs to make enzymes to catabolize lactose if lactose is present.
• When lactose enters the cell, allolactose, an isomer of lactose is formed.
• Allolactose binds to the repressor and alters its conformation so that it can't bind to the operator.
• RNA polymerase can now start transcription.
  • The three structural genes are transcribed to one mRNA strand.
  • The mRNA will then be translated into the enzymes that control lactose catabolism.
• In this sense, allolactose is an inducer.

Negative vs Positive Control

• While the Trp operon is an example of repressible gene regulation and the Lac operon is an example of inducible gene regulation, both are examples of negative control of genes because both operons are shut "off" by an active repressor.
• Gene regulation would be positive, on the other hand, if an activator molecule turned the operon "on".
• The Lac operon is also an example of a positive control system and is turned on by the cAMP-CAP complex, as the next section explains.
• E. coli can be described as a fussy eater.
  • Its first choice at every meal is glucose because glucose supplies maximum energy for growth.
  • Therefore, E. coli will only metabolize lactose if concentrations of glucose are low.
• For this to work, there must be a signal to tell the Lac operon that glucose is not available and to astart transcribing the genes to metabolize lactose.
  • This signal is a small molecule called cyclic AMP (cAMP).
    • The amount of cAMP present in a cell is inversely proportional to the amount of glucose present.
    • As a result, the absence of glucose results in an increase in cAMP in the cell.
• The following describes the situation where there is lactose but no glucose available to the cell:
  • No glucose means high levels of cAMP.
  • cAMP binds to a molecule known as CAP.
  • CAP, when in association with cAMP, can bind to the promoter at the CAP binding site.
  • Here, the cAMP-CAP complex stimulates transcription by helping RNA polymerase bind to the promoter.
    • RNA polymerase has a weak affinity for the Lac promoter and will not bind without this help.
  • Remember with lactose present so is allolactose.
    • Allolactose binds to the repressor and prevents it from binding to the operator.
    • Therefore, transcription and translation of the genes can occur.
  • The following depicts what happens when glucose and lactose are both present for E. coli to metabolize:
    • With glucose present, there is very little or no cAMP.
    • It cannot bind to the CAP binding site.
    • Without this complex, RNA polymerase cannot bind to the promoter and transcription cannot occur.
    • Even though allolactose is present and blocks the action of the repressor, there is no transcription of the lac genes because glucose is present.