GENE REGULATION AT THE PROMOTER LEVEL

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GENE REGULATION AT THE PROMOTER LEVEL

All cells use only a fraction of their total number of genes (their “genome’) at a given time. Gene expression is an expensive process, it takes a lot of energy to produce mRNA and protein and also a lot of often limiting nutrients such as N and P. It would be wasteful, for example, for a bacterium to produce a transport system for lactose and enzymes to metabolize it if there is no lactose in the medium. In fact, E.coli has three genes that are specifically required for lactose metabolism and these genes are only activated (“turned on”) when there is lactose present in the medium. In the absence of lactose the lactose genes are repressed by the binding of a repressor protein to the promoter. In the presence of lactose the repressor protein is removed from the promoter and the lactose genes are said to be de-repressed. The three genes required for lactose metabolism are assembled into a single unit, called an operon, under the control of a single promoter.

The preferred energy and carbon source for E. coli, as is the case for many other cells, the monosaccharide glucose. When gluocose is present genes specific for the metabolism for other carbon and energy sources, for example lactose, are incativated (turned off) at the promoter level. If E. coli is inoculated into a medium containing both glucose and lactose then the glucose is used first and the genes required for lactose metabolism are activated only when the glucose has been been used up. This type of growth, in which a favoured substrate must be used up before another one can be used by the cells is called diauxic growth.

Madigan and Martino (2006) “Brock: Biology of Microorganisms.”

In the above graph notice:

(1) The level of the enzyme called β-galactosidase begins to increase only when the glucose in the medium is exhausted. The enzyme is required to catalyze the the hydrolysis of lactose to the monosaccharides glucose and galactose.

(2) The growth rate on glucose is higher than it is on lactose. Glucose is clearly the “preferred” energy and carbon source for E. coli.

The metabolism of lactose requires the following specific enzymes. All the other enzymes for lactose metabolism are common to those required for glucose metabolism i.e. the enzymes of glycolysis and the Kreb’s cycle:

(1) lactose transport system (called lac permease) –actively tansports the lactose into cell

(2) β-galactosidase- catalyzes the hydrolysis of the disaccharide lactose to the monosaccharides glucose and galactose.

(3) Transacetylase- catalyzes the acetylation of β-galactosides such as lactose. Its function in lactose metabolism (if any) remains unknown, even though the structure of this enzyme has been determined. Mutants of the gene for this enzyme (the lacA gene) are unimpaired in terms of lactose uptake and metabolism.

Because there is only one promoter for the three contiguous genes there is only one mRNA produced. This mRNA is called a polycistronic message. The term “cistron” is an older term synonomous with “gene”. Each of the three messages in this mRNA message is translated individually because there is a ribosome binding site before each of the mRNA messages.

Notice that the polycistronic mRNA has three separate ribosome-binding sites, so that each of the three messages of the long mRNA molecule are translated separately.

Control of the lac operon

The diagram on the next page outlines how the lac operon is regulated.

The binding of

The binding of the lactose, at a specific lactose-binding site of the repressor protein causes a change in shape of the repressor protein so that it can no longer bind to at the operator site of the lac operon. The operator site could also be called the regulatory site. The roadblock to RNA polymerase binding to the promoter is thus overcome. The lactose is said to be an inducer of transcription.

So, if there is a requirement for intracellular lactose to “turn the gene on” how does this happen when the gene already needs to be turned so that the necessary lac permease cab be made to let lactose into the cell!?!. It turns out that there is some small amount of imperfection in the regulatory system that allows for a small “leakage” amount of transcription to always occur. This amount is so small that not much energy is wasted in making the small amounts of the three proteins (the permease, the galactosidase, and the transacetylase). Not much permease is required to catalyze the entry of the small amounts of intracellular lactose required for induction. Interestingly, it is not lactose itself that is the true inducer! The true inducer is allolactose, made from lactose by the galactosidase.

The above diagram and discussion describe how the presence of lactose is required for transcription of the lac operon. But it does not tell us why the presence of glucose obviously is able to over-ride the ability of lactose to induce the transcription of the lac operon (as is evident from the graph describing diauxic growth). The inhibition by glucose is an example of what is called catabolite repression. A catabolite is some product of the catabolism (breakdown) of a molecule. In this case the molecule being catabolized is glucose. The tern catabolite repression is actually mis-leading because it is not actually a breakdown product of the glucose that is involved in the inhibition but is actually due a certain molecule not being made during glucose catabolism!!. The molecule that is not made, or at least not in large amounts, is cyclic adenosine monophosphate (cAMP).

ATP cAMP + PPi catalyzed by adenylate cyclase

PPi 2 Pi catalyzed by phosphodiesterase

It turns out, then, that the [cAMP] inside E. coli is much lower when glucose is being metabolized than when other carbon substrates, such as lactose, are being metabolized. (we are not going to discuss why this is! )

departments.oxy.edu/.../102700/Fig_28_18.GIF

In this diagram we see that the operator (hidden by the DNA looping) is not the only regulatory site involved with the transcription of the lac operon. (The binding of the lac repressor to the operator region of the gene actually does cause the change in shape of the DNA strand seen in this diagram, but not in the previous diagram.) That other regulatory sequence is called the CAP site.

cAMP = cyclic AMP; CAP = cAMP activator protein; CAP site = DNA site where CAP binds

Here is how it all works:

(1) CAP only binds to the CAP site when cAMP is bound to CAP.

(2) The [cAMP] is only high in the absence of glucose.

(3) The higher the [cAMP] the more likely it is that all the CAP sites will be filled with the cAMP/CAP complex.

(4) In the presence of lactose the repressor protein does not bind to the operator.

(5) This means that RNA polymerase can bind to the promoter.

(6) When RNA is bound to the promoter it touches the cAMP/CAP complex (if it is bound to the CAP site).

(7) This touching causes an allosteric change in the RNA polymerase making its initiation of transcription more efficient.

The repressor protein is, of course, a repressor of transcription and the operator region of the DNA can be called a repressor region. The cAMP/CAP is an activator of transcription and the CAP site can be called an activator region.

TRANSCRIPTION UNIT (OPERON)

PROMOTER

OPERATOR

TERMINATOR

lacZ gene

lacYgene

lacA gene

lacZ mRNA

lacY mRNA

lacA mRNA

Transcription

Translation

DNA TEMPLATE STRAND

mRNA

lac permease

β-galactosidase

transacetylase

RNA POLYMERASE

Control of the lac operon.

Weaver (2005) “Molecular Biology”

[LACTOSE]

[GLUCOSE]

CAPcap

CAP sitecap