BIL 104 - Lecture 16

GENE EXPRESSION: The Link Between Genes and Proteins

We already know what PROTEINS are, and that they can be

structural (e.g., keratin of hair; silk)
functional (enzymes)
both structural and functional (e.g., muscle fiber proteins)

And we already know how proteins are manufactured from DNA by a stepwise process starting with

Transcription - the manufacture of RNA from the DNA template
Translation - the manufacture of polypeptide chains from the mRNA template

There is an endless variety of proteins and protein products--from blood-clotting factors to immune system elements to digestive enzymes--in living cells, each the product of genes.

How does the cell know which proteins to make, and when?

When a gene is actively being transcribed and translated into protein, we say that it is being EXPRESSED.

Obviously, not all genes are expressed constantly. Not only would this be wasteful, but it would not allow differentiation of cells and tissues during the development of multicellular organisms.

Hence, the cell must have mechanisms by which it can turn its genes "on" or "off", as necessary in its developmental cycle and according to its metabolic needs at any given moment.

To control the products manufactured by a cell at any given time in its development or life cycle, feedback mechanisms must operate which allow the cell to

2. induce the manufacture of a product not constantly made.

This control can be exerted at the level of

2. translation (i.e., protein not made from the mRNA transcript)

3. protein/enzyme function (enzyme can't attach to its substrate)

Inducible system - cell manufactures a product only when needed

Repressible system - cell usually manufactures a product, but shuts down production when the product isn't needed.

Also, a particular gene may operate under

Positive control - a protein called an "activator" attaches to the gene to allow transcription (the activator is something like an "on" switch)

Negative control - a protein called a "repressor" attaches to the gene to prevent transcription (the repressor is something like an "off" switch)

Let's have a LOOK.

Before we jump in, a couple of important definitions:

CATABOLISM: breaking down.
catabolic systems operate to allow the cell to BREAK DOWN a given substance.
ANABOLISM: building up
anabolic systems operate to allow the cell to BUILD a given substance.
THINK: catabolic and anabolic steroids (explain!).

Okay. Time to jump in.

For examples of gene expression control at the transcriptional level, we return to our old pal, Escherichia coli, your common intestinal bacteria. Right now, the E. coli in your intestines are busy making their own enzymes in order to help them digest the breakfast you so kindly tossed down to them a couple of hours ago. Let's have a look at what they're doing...

The lac operon - an inducible system
The trp operon - a repressible system

The lac operon:
An Inducible System that can operate under either positive or negative control.

First, a few terms...

operon

promoter - a region of DNA to which RNA polymerase attaches in preparation for transcription.

operator - a short DNA sequence, usually located just behind the promoter, to which a specific enzyme can attach.

The well-studied lac operon is the sequence of genes used by E. coli to produce three enzymes that allow it to digest the sugar LACTOSE.
These enzymes are:

catabolize lactose into glucose and galactose OR
isomerize lactose into allolactose (same chemical formula; different shape, as will be shown shortly).

2. Beta galactoside permease - which facilitates transfer of lactose into the cell and concentrates it in the cell

3. Beta galactoside acetyl transferase - this acetylates other galactoside sugars that may be in the cell (besides lactose), preventing Beta galacosidase from breaking them down (which can produce toxic side products and be wasteful--need to concentrate on lactose!)

E. coli doesn't always have lactose available as a snack. This means that it doesn't always "want" to be expressing the three enzymes that digest lactose.

SO HOW DOES THE CELL TURN THE LAC OPERON GENES "ON" AND "OFF?"

It's a rather amazing story...

The genes comprising the lac operon, and which code for the three enzymes, are known as "Z" (B-galactosidase gene), "Y" (permease gene) and "a" (transferase gene). All are under the control of the same promoter.

Separate from the genes of the lac operon is an INDEPENDENT gene, known as "i". It codes for a specific REPRESSOR PROTEIN which is capable of binding to the operator of the lac operon.

When the repressor is stuck to the lac operon's operator, RNA polymerase can't attach to the genes, and thus, cannot transcribe the mRNA needed to make those three enzymes.

Hence, when the repressor is attached, no lactose-digesting enzymes are produced!

BUT HOW DOES THE CELL KNOW WHEN THERE'S LACTOSE AROUND?

It becomes still more amazing...

The repressor is an allosteric (shape-changing) protein, which has a binding site for a MYSTERY MOLECULE, also known as an EFFECTOR (identity to be revealed shortly. Stay tuned.). When bound, the Mystery Molecule/Effector changes the binding properties of the repressor, causing it to release from the lac operator.

In the E. coli lac operon, the inducer (mystery molecule) is an isomer (i.e., same chemical, but with a different three-dimensional shape) of lactose....ALLOLACTOSE.

When E. coli is in the presence of milk sugars (lactose), the following events occur...

2. The allolactose binds to the allosteric binding site of the repressor.

3. When allolactose is stuck to the repressor protein, the protein loses its affinity for the operator/promoter of the lac operon, and RELEASES from it.

4. RNA polymerase (remember this enzyme? It's the one that builds mRNA directly from the DNA template during transcription) can now attach to the naked promoter, and transcribe the three genes, Z, Y, and a.

5. The new mRNA transcripts from the operon are then translated into the three enzymes that help the E. coli cell digest (i.e., catabolize) lactose.

This system operates under POSITIVE FEEDBACK: as long as there is lactose in the cell, Beta galactosidase will continue isomerizing some of it into allolactose.

When allolactose is present, it binds to the repressor proteins, keeping them OFF the operator of the lac operon.

Thus, transcription continues as long as there's enough lactose to keep the repressors ("off" switches) away from the lac operon.

How does the operon turn off when the cell runs out of lactose to digest?

2. Following the new concentration gradient of allolactose, any allolactose molecules still bound to the allosteric repressor molecules diffuse off.

3. Beta-galactosidase can now split these released allolactose molecules and use them for energy, too.

4. Meanwhile, the unbound repressor protein returns to the lac operator and clamps back on. 5. The attachment of the repressor protein TURNS OFF transcription of the lac operon.

6. The three enzymes used to digest lactose are thus no longer being made by the cell!

Once transcription and translation of the operon has ceased, some of the manufactured enzymes will still remain in the cell for a while, though most will eventually be degraded/inactivated. Any remaining Beta-galactosidase molecules (which actually last for a relatively long time) may be instrumental in the initial

lactose-->allolactose

isomerization upon the E. coli's next lactose meal.

How was the operon studied? You guessed it. With Mutants who have FLAWS in any of three enzymes made by the lac operon. Once again, we can best learn about how normal genes operate by studying ones that are faulty.

The lac operon is the classic example of an INDUCIBLE SYSTEM. But there are other types of systems, including, equally famous...

The trp operon:
A Repressible System The tryptophan (trp) operon is a REPRESSIBLE system. (AAAUGH! Scary diagram alert! But don't worry, I will explain the diagram step-by-step.)

TRYPTOPHAN is an amino acid, and it's generally needed by the cell all the time, to make proteins of various types. Under normal circumstances--the cell always needs to be making tryptophan.

1. To make tryptophan, E. coli needs the five specific protein products of the five trp genes (shown in the diagram), each of which drives one of the reactions necessary to change a precursor (chorismic acid) into its final product, TRYPTOPHAN.

(Don't even think about memorizing the diagram. But do understand what's going on in the picture. OVERALL: Chorismic acid is making a stepwise transformation into Tryptophan, with each change mediated by an enzyme encoded by the trp operon.)

2. At a completely different locus from the trp operon, there is another gene (oh no...) NOT included in the trp operon shown in the diagram. It's not part of the trp operon; this gene's name is trpR.

3. The protein product of the trpR gene, a REGULATOR PROTEIN, is inactive under normal circumstances (i.e., when the cell needs tryptophan, which is almost always).

4. However, when there's an excess of tryptophan in the cell, some of that excess tryptophan will bind to the REGULATOR PROTEIN, activating it.

5. This activated REGULATOR-TRP COMPLEX binds to the trp operator, blocking RNA polymerase from attaching and transcribing. 6. The trp operon is thus turned "off": the enzymes that build (anabolize) tryptophan are not manufactured by the cell.

Because the very product of the operon (tryptophan) is involved in turning off the genes that allow the cell to make it, tryptophan is known as a COREPRESSOR.

What happens when the cell needs tryptophan again? How does it turn the operon back "on"? 1. When the cell's trp concentration becomes very low, tryptophan will diffuse off the regulator protein.

2. This changes the shape of the protein, and it loses its affinity for the trp operator.

3. The regulator, naked of its tryptophan corepressor, releases from the trp operator.

4. With the regulator gone, RNA polymerase can now attach to the operon and transcribe the five genes coding for the five enzymes that built tryptophan out of chorismic acid.

In the above condition, the operon is said to be DEREPRESSED until tryptophan concentration becomes high enough to turn off the gene again.

It's like magic.