Friday, 8 March 2013

Operons! Elegant ... beautiful ... obvious.

This is a tricky section for a lot of reasons.  First, it's critical that students understand the functions of all the parts of genes.  The promoter is, of course, where RNA polymerase binds (see previous lectures).  The polymerase makes RNA.  In the case of gene regulation, it's reasonable to assume that the RNA created is mRNA.  Prokaryotes can also take advantage of polycistronic mRNA - multiple reading frames in a single molecule of mRNA.  Ribosomes latch on with a Shine-Dalgarno sequence, and move from 5' to 3', making polypeptides.

Knowing that, think of the possibilities!  If you control the mRNA, you control how much protein can be made.  This is a good example of gene regulation.

Making molecules is expensive

For prokaryotes, it's most efficient to control gene regulation by stopping transcription.  If you don't make RNA for a protein, you save a lot of cellular energy.  Each nucleotide consumes one ATP equivalent (the ATP, GTP, CTP and UTP are all similar in energy profiles).  And then there's the maintenance!  It takes more energy to make an ATP than you get out of it.  Making RNA is expensive.  You might think ALL cells should use this type of regulation!

Cells are like cities - particularly eukaryotic cells

Keep in mind that it's also expensive to make a polypeptide.  GTP pushes the ribosome along the mRNA.  ATP charges each tRNA with its appropriate amino acid.  Making the amino acids takes energy, as does recycling of unused proteins (and of course their polypeptides).  Even if you use post-transcriptional regulation (you regulate even having MADE the polypeptide), you can still save some energy.

The bottom line is:  Don't make things you don't need.

Why not just use transcriptional regulation?  Let's look at how Eukaryotes often have to manage their multicellular situations.  You might have a tissue that has the job of secreting a hormone to signal a dramatic change with little warning.  You can't do this well with transcriptional regulation.

I sometimes make an analogy of the cell being like a small city.  The nucleus can be city hall:  it contains plans and directions for growth and controlling how things get done.  The mitochondria are the powerhouses.  Lysosomes are the custodians, breaking down and cleaning up outdated or unnecessary parts.  What about the police?  Or the fire department?  You don't want to start recruiting, training, and equipping either of these on short notice.  If you start to look for people who would make good firemen when there's a building that's just started on fire, you'd be months late to do anything about it.  Post-translation processing - like what happens with insulin - make the response time dramatically shorter.

You can see why it might be useful to have different levels of gene control at hand.

Back to Bacteria

This section is about how prokaryotes do something eukaryotes do not:  they use operons.  I think that if I do my job as an educator well, you'll be disappointed that you, as a eukaryote, don't do this yourself.  Operons are like a chandelier.  You turn it on to impress people.  You don't turn on HALF the bulbs ... you turn them all on.  You have one light switch, which is elegant, beautiful, and obvious.  You wouldn't have one switch for every bulb in the chandelier.

Can I throw another analogy at you?  This one is an assembly line.  Each job is done by one worker.  A biochemical pathway is like that.  Each enzyme is, by nature, able to do only one job.  If you are taking some reactant and turning it into a product in four steps, you ALWAYS need all four enzymes.  You can never "make do" with just three.  If one worker is sick one day, the other three will only cost the company money in wages for work that can't get completed.

If you put all the genes on a single mRNA, you get all the workers.  You don't even need the workers arranged in a particular order:  you just need them linked by an "on switch".  For RNA (i.e. transcriptional control) that "on switch" is the promoter.
Note that you get four DIFFERENT polypeptides from the same mRNA strand.  They have different amino acid sequences, and therefore different functions.

Enter the operator

The operator comes between the promoter (or is a part of the promoter) and the +1 nucleotide in the mRNA. Such an arrangement makes up an "operon".  The operator can bind to a protein to prevent synthesis of the mRNA.  This is negative regulation, and you'll see more about it in the video that follows.

The protein that binds to the operator is called the "repressor".  It can sense whether you want to transcribe the operon or not.  The way it senses the conditions is by having two important sites:  one site is the DNA binding domain, which is where it contacts the DNA.  The other site is where it binds to some other molecule.  Binding the other molecule changes the shape of the repressor.  One conformation is that the repressor can bind DNA at the operator.  The other conformation means it cannot.  This shape alteration is called an allosteric shape change.

To see how this works in practice, watch the video lecture below:

Don't always be so negative.
There's also positive regulation.  The bacterium has a vested interest of making the best use of its resources.  Sure, it's great to have the ability to break down lactose, but what are the end products?  Glucose and galactose - and the latter is an isomer of the former.  Galactose is quickly converted into glucose, and then is processed through glycolysis.  So, does it make sense to make the enzymes to break down lactose if you already have glucose?  Remember - making enzymes involves transcription and then translation, which are energetically expensive!

The answer is no.  And the cool thing - to me - is that bacteria figured this out by themselves, and they don't even have a brain or reasoning power!  Man, but evolution is cool.

Bacteria sense the amount of glucose by watching the product of another chemical reaction.  The enzyme adenylate cyclase converts ATP into cAMP (cyclic adenosine monophosphate).  The cAMP acts as a messenger to the cell ... it says "I'm starving".  How does it do that?  It turns out that when glucose levels are sufficient, adenylate cyclase activity is inhibited.  Glucose interferes with the production of cAMP.  Thus, when glucose is high, cAMP is low and vice-versa.  cAMP activates the protein CAP (sometimes called CRP, for "cAMP Receptor Protein").  When CAP-cAMP binds to the promoter of the lac operon, it revs up transcription to a very high level.  If lactose is absent, repression prevents transcription ... but if lactose is present, the receptor protein is deactivated and you get tonnes of permease and beta-galactosidase (and transacetylase, whatever that's for ...).

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