Gene Regulation

Need to regulate expression - "waste not". Enzymatic activity and transcription/translation are energy consuming processes. Generally, bacteria do not expend energy unnecessarily.

How do bacteria regulate gene expression?
1. control the activity of preexisting enzymes
2. Control transcription/translation

Controlling activity of enzymes - this approach allows cells to respond rapidly to their environment.

Product inhibition - enzymes participate in reactions that can go in either direction. If the concentration of the product builds up the activity of the enzyme may be reversed from product to substrate.

Feedback inhibition - usually occurs in pathways from substrate A to product F. Product F may inhibit some key enzyme near the beginning of the pathway. Again, as the concentration of the product builds up, the pathway is inhibited.

How? The key enzyme is an allosteric enzyme with two important sites: the active site and the allosteric site. The allosteric site binds reversibly an effector molecule (e.g., product) that causes a conformational change in the active site so that the substrate can no longer enter the active site effectively.

Transcriptional/translational control or regulating protein synthesis.
simplest of them

Enzyme repression - an endproduct of a pathway represses the synthesis of the enzymes. Found in the biosynthesis of amino acids and nucleotides. The substance that represses transcription is called a corepressor. The corepessor binds to a repressor protein, an allosteric protein, and causes a conformational change in the repressor such that it binds to the operator region, a region close to the promoter region.

Enzyme inductions - synthesis of the enzymes only if substrate is available. Frequently associated with catabolism of carbon and energy sources. The substance that induces transcription is called an inducer. A repressor protein binds to the operator and blocks transcription when the inducer is absent. When the inducer is present and binds to the repressor, the repressor cannot bind to the operator and transcription proceeds. Notice the underlying mechanism is for the repressor to inhibit transcription and the repressor activity is effected by a small molecule. Inhibition by a repressor in general is called negative control.

In both cases they work on the need basis! don’t need repress; do need induce!

Constitutive enzymes are enzymes that have little or no control. Their synthesis is pretty much level throughout the life cycle of the cell. These are house hold type enzymes that are required for the daily maintenance of the cell.

What if a bacteria is faced with two different carbon sources, e.g., glucose and lactose? Perhaps one carbon source is better than the other. What would a bacteria do? Not express the genes necessary to metabolize the poorer carbon source is a good idea? Repress these genes somehow.

Exactly what we will look at - a phenomenon called catabolite repression.

Catabolite repression or Glucose effect - is defined where a simple energy source is preferentially used over a more complex energy source. The utilization of the one energy source, in this case glucose, represses the genes of many unrelated enzymes.

What does the phenomenon look like?

One consequence is what is called diauxic growth. In diauxic growth the organism grows on the preferred substrate and then switches to metabolize the alternative substrate.

For example inoculate a flask of medium containing glucose and lactose with Escherichia coli. E. coli will grow on the glucose until it runs out of glucose. There will be a brief cessation of growth - a lag period- and then the cells will grow on lactose as a carbon and energy source.

Operon model for gene expression - Lac operon model from Francois Jacob and Jacques Monod. Operon consists of a promoter, operator and the structural genes in this case lac z, y and a which code for beta galactosidase, a permease and transacetylase respectively. Near the operon is a gene called lacI which codes for a repressor for the lac operon.

When lactose is absent, the repressor molecule binds to the operator region and nearly completely shuts down transcription of the the operon by RNA polymerase. This is analogous to the inducible operon we talked about earlier.

When lactose is present, some of the lactose is converted to allolactose, the inducer of the operon. Allolactose binds to the repressor and inactivates it such that it will not bind to the operator region. Therefore, RNA polymerase can transcribe the operon.

What happens if glucose and lactose are both present in the medium?

What was happening during growth on glucose? - Catabolite repression. Catabolite repressed operon require an allosteric protein called catabolite activator protein or CAP to bind to the DNA before RNA polymerase will bind and transcription will occur. CAP will bind to the CAP binding site of the DNA if and only if it has bound cyclic AMP. In the absence of cAMP, CAP will not bind to catabolite repressed operons. Glucose inhibits the synthesis of cAMP in a complex manner.

So what is happening in our culture of E. coli? The presence of glucose is keeping the levels of cAMP low, which does not allow CAP to bind to the lactose operon and therefore the lac genes are not transcribed or translated. There are many other operons that are controlled by cAMP levels and CAP.

What then is required for the lac operon to be expressed?

1. cAMP bound to CAP which binds to the CAP binding site to activate transcription of the operon.

2. There must be an inducer such as allolactose, which binds to the repressor and prevents it from binding to the operator region. An example of negative control since there is a repressor regulating transcription.