Friday, 16 March 2018

Elucidating Codons with Random Copolymers

Before we got a codon table as we know it today, scientists had to figure out what the letters were in each of the 64 combinations of triplets of nucleotides.  It is child's play to get all the possible codons, but what they mean is another story!

The level of technology at the time was that random polymerization in vitro (that is, in a testtube with enzymes, buffers, and whatever nucleotides the scientist put into the tube) was possible, but precise construction of synthetic mRNAs wasn't an option.

So, scientists created synthetic mRNAs that contained only, say, uracil and cytosine.  To distinguish between U and C content of each codon, they ensured that the proportion of U would not equal C.  Thus, they knew if 80% C and 20% U were used, the most common codon would be CCC and the least common would be UUU.  The number of CCC would be 0.83 and UUU would be 0.23.

Using this logic, answer the following question:

  • A scientist creates a random copolymer from a solution containing all 20 amino acids, 40% guanine, 60% cytosine, and appropriate enzymes.

  • She then does in vitro translation and isolates only the oligopeptides that were formed.

  • Your job is to calculate which amino acids were in the polypeptides formed and the proportion of each represented.

Try this for yourself before accessing the answer below ...

Tuesday, 12 November 2013

Bidirectional Replication

Here's a quick video (~12 min) regarding the replication fork and DNA synthesis.   It is done by Dr. Bird.

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 ...).

Saturday, 2 March 2013

Translation of Genes

Now that we've got transcription under our belts, we can now look at translation.  Translation is where the information that was stored in genes (DNA) is used to create a protein.  Note that the DNA information had to be copied (transcribed) into a new molecule: RNA.  It's very common for students to be unclear about what processes occur during transcription and which ones are translation.

When you're talking about promoters and terminators, enhancers, silencers, and RNA polymerase, you're talking about transcription.  In eukaryotes, RNA splicing, mG capping, and the poly-A tail are all involved in modifications of the transcript (RNA strand).  These events occur BEFORE translation.

Note that only when we're dealing with translation do we start to worry about start codon and stop codons.  Most people have dealt with codons and anticodons in their introductory biology classes, but in my lecture below I look them over again quickly.  Note that I'm adding a little more complexity to these introductory concepts:  I want everyone to know that there's special parts of mRNA - the 5'UTR and the 3'UTR (untranslated regions).  The borders of these are defined by the start and stop codons.

In translation, there's also a set of steps to getting the ribosome to load unto the mRNA.  The ribosome is made of rRNA and protein.  The small subunit in prokaryotes has a piece of rRNA that's complementary to the 5'UTR upstream of the start codon:  the Shine-Dalgarno box.  Eukaryotes have something similar (the Kozak sequence, but which has the start codon embedded within the consensus sequence).  After the small subunit has bound, the first tRNA with its amino acid (methionine in eukaryotes, formylmethionine in prokaryotes) will bind to the start codon, then the large subunit attaches.

Prokaryotes also transcribe and translate their genes simultaneously.  Without a nuclear envelope to partition the RNA polymerase in one compartment (the nucleus) and the ribosomes in another (the cytoplasm) we can see transcription of mRNA that occurs even as it's being manufactured!  Eukaryotes have spacial separation, which may be why they are able to modify their mRNA so extensively before translation.

So, without more buildup, here are the video lectures for "translation"!

Part 1:  Structure of the ribosome

Part 2:  Using the Code

Part 3:  Cracking the Code

Part 4:  Posttranslational Modification and Shipping

Thursday, 21 February 2013

Transcription of Genes

I'm going to try something a bit new:  I'll make the videos that usually accompany my genetics class with copyright-free images and provide the theory and context of genetics in a way that I can post online outside of our course management system.

So here's the first-ever fully public presentation of "Transcription", which I'll do in two parts!
Part 1 - Overview

Part 2 - A little more detail

Wednesday, 13 February 2013

How cool is that structure?

Today in class we were to go over the structure of DNA.  The structure is intimately linked to its function, so the base-pairing aspect is HUGE.  The fact that a string of bases on one DNA molecule can dictate the order of nucleotides on the partner molecule is the only thing that allows information to be transferred from cell-to-cell during division, and by extension from parents to offspring generationally.

To show the awesome beauty of the molecule I showed a great site in class: This was put together by Dr. Eric Martz and he lets people use it for free.  I think being able to rotate and move the molecule, to zoom in, isolate nucleotide pairs, and all the features here are a great way to really appreciate how DNA works!

I encourage you to go to the site yourself and see what you can see.  The link I gave above is the hands-on part.  I thought I'd share with you my thoughts about this activity in a little movie (below).

Have fun!

Monday, 10 December 2012

A Fusion Operon Question

What's a fusion operon?  Ask Dr. David Bird, an evil scientist who likes to test the understanding of his students regarding gene regulation by creating unholy and absurd mixtures of operons.  Well, that's overstating it, but it's a neat puzzle that is created by joining control structures from different operons together.

In this case, the CAP site and operator of a lac operon were fused to the trpL and structural genes of a trp operon.  Remember that the CAP site is for positive regulation, the lacO is bound by a repressor protein (it's not in this figure, but imagine it's there) in the absence of lactose.  The trpL is the leader sequence that allows attenuation (premature end of transcription unless tryptophan levels are really low - it does this by making a hairpin structure that results in rho-independent termination of transcription).

See if you can fill out this table using those cues.  Oh, and thanks go to Dr. Bird for his generously supplying me with this example problem (and Brittany for reminding me about this exercise)!

The solution: