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More on the Lac Operon

A while ago I wrote two posts about the Lac Operon here. The first pointed to an animation by McGraw Hill Publishers that did a pretty good job illustrating how the operon works. In the second post, I highlighted the notion of polycistronic messages (more than one gene per mRNA molecule) and how this allows for control of a number of related genes at once – a trait not shared by eukaryotic cells. In that second post, I also finished with a graph of how cells grow in the presence of glucose and lactose.

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Cell Growth in the presence of glucose + lactose – As glucose is depleted, cells adjust to lactose digestion

One feature of that graph (reproduced here) that is notable is a little bump in the growth rate as glucose runs out and the cell converts to lactose digestion. A second important feature is that the rate of growth slows when the cell is burning lactose as its primary fuel.

 

Together, these features suggest that the cell is regulating lactose digestion very closely. In fact, there are two primary mechanisms of this regulation to appreciate. The first is that the lactose-digesting enzymes are controlled together on an operon that is regulated by lactose itself (or at least we can assume so for simplicity’s sake). In the absence of lactose, no lactase enzymes are made and no lactose is used as fuel. The reason for this is obvious when you look at the slope of cell growth under glucose metabolism (left) and lactose metabolism (right). Clearly, growth is SLOWER when lactose is used as fuel.

Therefore, so long as there is glucose, it is pointless to digest lactose at the same time. So it is best to only turn on the lac operon in the ABSENCE of glucose – regardless of whether lactose is present of not.

If glucose is absent and lactose is absent, turning on lactase enzymes is still useless. However, slow growth is better than no growth. So we should have a mechanism to turn on the operon when there is lactose in the environment.

Here’s a matrix of ideal regulation:

Screen Shot 2014-03-31 at 2.18.05 PM

How can a little, mindless bacteria achieve this exquisite control?

Simple: By using two regulators. One for glucose and one for lactose. Only when both conditions (glucose-, lactose+) are met do we make lactase.

Structure of the Lac Operon

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First, lactose itself serves as an inducer. In the absence of lactose, a regulator protein binds to a DNA site between the polymerase binding site (the promoter) and the structural genes (the enzymes). When the regulator binds, its presence physically prevents the progress of RNA Polymerase.

When lactose is present, it binds the repressor protein in a way that causes its shape to change in a way that can no longer bind the DNA. The repressor then drifts away from its binding site allowing RNA Polymerase a clear shot to the structural genes.

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However, RNA Polymerase is not always parked on the promoter waiting for the repressor to be removed. Its binding requires another protein to help stabilize its interaction with the DNA. This second protein is the CAP protein. The Catabolite Activated Protein. However, CAP alone will not bind either. It requires a signaling molecule called cyclic AMP (cAMP). cAMP is readily broken down when glucose is in the cell, so it only accumulates when glucose is absent. In that case, cAMP accumulates and binds to the CAP protein, which then binds to the CAP site. This site is located adjacent to the promoter, but on the side away from the structural genes. When CAP binds, it assists in recruiting the RNA polymerase to the promoter.

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Therefore, if only one condition is met, it is insufficient to promote gene transcription. Only when the CAP+ cAMP protein is bound will the Polymerase be recruited. And only when lactose is present, will the repressor protein let the Polymerase pass.

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In terms of the matrix we set forth above, we can see that these molecular interactions result in exactly the regulation that is optimal:

post-operon

 

 
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Posted by on March 31, 2014 in Uncategorized

 

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A quick followup on that lac operon post

Last week I posted a quick link about operons for my micro class to check out before taking their quiz on bacterial gene regulation This post is intended to complement that one. To go back to that post, click here. If there’s one thing to remember about operons it is that bacteria, lacking a nuclear membrane, regulate their genes differently than Eukaryotes. Having a nuclear membrane separates transcription and translation into two distinct compartments allowing for more subtle tweaking of Eukaryotic mRNAs before they are exported for translation.

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Click on this figure to go to a good description of how polycistronic genes work

One thing this does is it makes it very beneficial to package genes with related function closely on the genome and use a single regulatory region to control them all together. They wind up getting packed so closely together that they are actually expressed as a single messenger RNA – known as a polycistronic (meaning ‘many gene’) message.

Upstream of this polycistronic cassette are regulatory elements. One element common to all regulatory elements is the promoter. The promoter consists of several elements which ‘promote’ the binding of an RNA polymerase to the DNA. Additional regulatory elements exist to ensure that this polymerase only transcribes the genes if they are needed. In doing so, the cell conserves energy and components (e.g. Amino Acids) for only necessary processes.

In the case of the paradigm lac operon, lactose is a fuel source, but not as good as glucose. Therefore, enzymes to digest lactose are only needed when lactose is present, but glucose is not. In order to interrogate both conditions, two additional regulatory elements are present.

First, the operator sequence. This sequence binds a repressor protein that physically blocks the polymerase’s path in the absence of lactose. However, if lactose is present, the sugar binds to the repressor, causing a conformational (shape) change that causes the protein to release its grip on the operator sequence.

Second, a catabolite activator protein (CAP) will only bind to the DNA behind the RNA polymerase if cAMP is present. Let’s not get too distracted, other than to say that cAMP levels are high in the ABSENCE of glucose, and low when that sugar is present. When cAMP binds to the CAP protein it can now bind the DNA and do it’s other job: making a nice binding site for the RNA polymerase. Without CAP, the polymerase binds very inefficiently.

Together, the production of lactase enzymes (those that digest lactose) is exquisitely controlled in a way that conserves the most energy.

ImagePs – take a look at this graph and tell me why (not mechanistically, but rationally) the cell does not make lactase enzymes when both glucose and lactose are present.

 
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Posted by on March 15, 2014 in Uncategorized

 

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Lac Operon

I’m typically not a fan of McGraw-Hill’s animations because they are rather dry and un-engaging, however, this one does a good job of describing conditions under which the lac operon will be either ‘on’ or ‘off’ and illustrating how positive and negative regulatory elements work together so that the operon is on when needed, but conserves energy by remaining off when it is not needed.

http://highered.mcgraw-hill.com/sites/0072556781/student_view0/chapter12/animation_quiz_4.html

more on this later….

 
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Posted by on March 10, 2014 in Uncategorized

 

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