The Trp Operon

The last post about an operon (the lac operon) is the most viewed post on this blog, so I thought that it might be helpful to follow this up with another operon, this time concentrating on the trp (tryptophan) operon.  This operon is another really elegant example of transcriptional regulation in E.coli and the mechanism is pretty cool.

Amino acids are essential for life (see the last post on their composition!) and cells synthesize amino acids using a variety of enzymes.  When nutrients are plentiful, such as E.coli would encounter in nutrient broth in the laboratory setting, cells no longer need to waste energy producing biosynthetic enzymes when they can utilize nutrients already in excess.  The trp operon contains several enzymes that are coordinately regulated and involved in the production of tryptophan.  When tryptophan is present in the cell's environment, it doesn't need to make any of these enzymes, but if the cell needs tryptophan, these enzymes are transcribed and shortly thereafter translated.  Control of this operon, thus, controls how much energy the cell is going to put into making tryptophan.

Similar to the lac operon, the trp operon contains an operator (O) sequence, within the promoter sequence, where an operator binds and prevents transcription.  In the presence of tryptophan, the operator binds the promoter and prevents RNA polymerase from transcribing genes.  In the absence of tryptophan, however, transcription occurs at a basal rate.  Sounds simple enough, right?  Let's take it a step further and consider...

Attenuation
An important concept in gene regulation is that of attenuation, which is fine-tuning of gene expression.  You might think that attenuation is mediated by protein factors that bind the DNA and affect gene expression; however, attenuation of the trp operon is a little different and, instead, depends on mRNA structure to modulate gene expression.

Before moving forward, let's look at the trp operon (diagrammed to the right).  Briefly,t here are four regions, and these four regions have differing levels of complementarity to each other.  Thus, when the DNA is transcribed into mRNA, the mRNA folds into all kinds of shapes and the regions of the trp operon fold on each other.

After transcription of the entire trp operon (we're dealing with mRNA from this point forward), the next event is translation of this mRNA into protein.  In bacteria, it's important to note that transcription and translation occur simultaneously, so as soon as we have a transcript in a bacterial cell, it's being translated.  The trp transcript contains two critical tryptophan codons immediately before region 1, so in order to synthesize the enzymatic machinery to make tryptophan, the cell must use a few residues to translate the protein.

In the presence of high amounts of tryptophan within the cell, the ribosome plows through these two tryptophan codons, adding in the appropriate amino acids, and continuing through region 1 of the mRNA.  This results in region 1 and 2 mRNA sequences binding together, and then regions 3 and 4 bind together as well.  This interaction between regions 3 and 4 results in the creation of a transcription-termination hairpin, basically a structure in the mRNA that kicks out RNA polymerase and prevents further transcription of the mRNA.  Thus, transcription (and then translation) are stopped because

In the absence of tryptophan, however, the ribosome cannot quickly add tryptophan during the translation process and it stalls before region 1.  This results in the folding of the mRNA such that regions 2 and 3 bind to each other.  When this structure forms, no transcriptional termination hairpin is formed, and mRNA synthesis continues.  Thus, the entire mRNA sequence for the trp operon is made and can be translated into enzymes that will synthesize tryptophan.

In summary:
Lots of tryptophan: Ribosome zooms through the mRNA, regions 1 & 2 and 3 & 4 bind (in pairs) and create a termination hairpin
End result: Transcription terminates and tryptophan synthetic enzymes not created (cell saves energy!)

Lack of tryptophan: Ribosome stalls immediately before region 1, regions 2 and 3 bind each other, no termination hairpin is formed
End result: Transcription continues and biosynthetic enzymes are eventually synthesized

This scheme is similar for other operons encoding amino acid biosynthetic enzymes (in bacteria, that is).  The trp operon is an elegant scheme to finely-tune transcription via mRNA structure to prevent the cell from wasting energy.

Amino Acids: The Building Blocks of Proteins

While I've written many posts describing signaling pathways and cellular phenomena of significant complexity, I'd like to use this post to take a step back and look at some fundamental building blocks, first turning to amino acids, the monomers that, when polymerized, make up polypeptides and proteins. At the most basic level, amino acids are really a rather simple chemical compound, consisting of a amino group, a carboxy group, a hydrogen, and a side chain, all sticking off a central carbon atom.  These amino acids are polymerized via their amino and carboxy chemical groups to create long, linear linkages.

Brief aside: In my chemistry class in undergrad, my TA helped us remember the order of the chemical bonds following polymerization by saying N-H, C-H, C-O, N-H, C-H, C-O, ...


You may remember briefly from any stint in chemistry class that a carbon atom that is covalently bound to four different chemical entities (in this case, a side chain, a hydrogen atom, a carboxyl group, and an amino group) can take two different conformations, depending on how these bonds are spatially oriented.  In the case of amino acids, the vast majority of amino acids found in our bodies and used to generate proteins are L stereoisomers.  This is a result of the amino acid synthesis machinery structure exclusively generating L amino acids.  There are exceptions, but we won't get into that.

As I mentioned, amino acids have a side chain: the part of the amino acid that endows it with its identity.  These side chains can be broken into a few groups that we will explore now:

The first set of side chains is the nonpolar, hydrophobic side chains.  The amino acids in this group include alanine, valine, leucine, isoleucine, glycine, methionine, and proline (structures shown to the right).  What you'll immediately notice is that these amino acid side chains are composed mostly of hydrogens and carbons.  Thus, these side chains do not contain polar covalent bonds and do not interact as readily with water (thus the term hydrophobic - they're "afraid" of water.  Some amino acids of note in this group are proline, which contains a ring structure that creates a "kink" in the amino acid, and methionine, which contains a sulfur atom.

The next set is composed of the aromatic side chains, which includes phenylalanine, tyrosine, and tryptophan.  These amino acids all contain an aromatic ring, which makes them relatively nonpolar; thus, they do not interact favorably with water.  These amino acids are involved in mediating protein protein interactions and are frequently found at the active sites of enzymes.

Next up: polar, uncharged side chains: asparagine, cysteine, glutamine, serine, and threonine.  These amino acids contain hydroxyl, sulfhydryl, or amide groups that mediate interactions with water, but they carry no net charge.  An amino acid of note in this family is cysteine, which can react with itself to form cystine, which is important in mediating the formation of disulfide bonds in protein structures.

We'll consider basic side chains next.  These amino acids consist of arginine, histidine, and lysine, which all carry a net positive charge in solution.  Of note, histidine is commonly found at the active site of enzymes to serve as a protein donor or acceptor.

Finally, we find acidic side chains: aspartate and glutamate.  In solution, these amino acids carry a negative charge and are considered acidic.

In the diagram at right, I've drawn up each of the amino acids along with their three-letter and one-letter codes.  These codes are frequently used to abbreviate long lists of amino acids.

Another brief aside:  Did you know that a single woman designated the amino acid abbreviations?  She chose letters that made sense for most amino acids (as you can see above).  For tryptophan, for example, she chose W because she envisioned saying tryptophan as twyptophan.  Kind of cool, huh?


As a summary, here are the amino acid abbreviations:

• A, ala, alanine
• C, cys, cysteine
• D, asp, aspartate
• E, glu, glutamate
• F, phe, phenylalanine
• G, gly, glycine
• H, his, histidine
• I, ile, isoleucine
• K, lys, lysine
• L, leu, leucine
• M, met, methionine
• N, asn, asparagine
• P, pro, proline
• Q, gln, glutamine
• R, arg, arginine (think aRRRRginine)
• S, ser, serine
• T, thr, threonine
• V, val, valine
• W, trp, tryptophan (tWWWyptophan)
• Y, tyr, tyrosine

So there you have it: 20 amino acids.  In addition to these amino acids, our bodies contain several more, including selenocysteine (identical to cysteine but containing selenium rather than sulfur) and ornithine (remember this from glycolysis?).  Amino acids can also undergo modifications: for instance, lysine residues can be acetylated.  More amino acids and their variants are always being discovered as well.

Now that we have the building blocks of proteins established, the next blog post will focus on how these amino acids can be combined (polymerized) into long structures that make up polypeptides and proteins.

Android Applications for Scientists (and other people too!)

If you're like me, you like to use your phone for its capabilities, including for work, and because it's shiny and you paid a lot of money for it.

I use an Android phone (no thank you, Apple) and am always interested in an application that could help me with my research or studying.  Unfortunately, the Android marketplace is cluttered with irrelevant applications, making finding useful applications difficult.

The following applications are presented as a summary of what I use.  Certainly there are more out there (please tell me!) and more are created every day, and I look forward to using these applications in the future:

Astrid Tasks:  Every morning as I'm walking into the building, I fill out my task list of things to do for the day.  I don't need anything fancy - I just need an application that is quick, easy, and simple.  Astrid is fantastic for putting together this list and for prioritizing my experiments and work.  Plus, it's got a handy widget (if you're into widgets, that is).  I would highly recommend this application for those who like to keep lists.

GoogleDocs: If you use Google for composing, sharing, or viewing documents, this application is fantastic because it will sync your computer and your phone to view the same documents.  Additionally, if you've got a big enough screen, it's not too horrible to actually compose in these documents.  I don't use this too often, but it's convenient  when I've got a document to get on my phone and my Dropbox happens to be full (see below).

Doodle:  Have you ever tried to schedule a meeting with faculty?  How about with multiple faculty?  Needless to say, it is a nightmare: herding cats as many would say.  Doodle attempts to make this a little bit easier by creating a spreadsheet which participants can then check off for their ability.  That one magical time spot that everyone checks is then the meeting time.  Doodle comes with several options for creating an event and then adding participants.  In my department, these things are really the best way to make sure that a meeting is really going to happen.

Dropbox:  If you're not already on Dropbox, seriously, sign up for it.  This program is the most useful thing I have every used.  Once installed on a few computers and on your phone, a folder is created - your Dropbox.  It acts just like a normal system folder and can be manipulated just like one too.  The great thing is that anything that is in your Dropbox can be accessed on any computer that has Dropbox installed and you can also access your files online.  This is so much better than carrying around a flash drive, and the syncing is instantaneous.  I could not recommend this program more.

handyCalc:  Usually, I'll use my crappy old calculator from high school to do the simple arithmetic needed for my experiments and notebook.  When I'm at my computer and can't find my calculator, I use handyCalc, mainly because I find it much easier to use than the standard calculator that came with my phone.  There are multiple iterations of calculators out there - from simple to mind-bogglingly complex.  This program will solve equations, create graphs, and perform simple addition and subtraction.  It works and I like it.

LinkedIn:  At a recent conference, I learned the importance of LinkedIn.  The seminar speaker asked everyone in the room to raise their hand if they were a member of LinkedIn.  I was the only person not to raise my hand.  LinkedIn is a Facebook for professionals - it can be helpful if you're looking for a job or want to make some contacts to look for a job in the future.  Being able to use it on my phone is convenient, too.

Pulse:  I like to read the news on my phone when I can't access a computer.  The best app I've found for this is the Pulse reader app.  Using this program, you can view tiles containing the top headlines from various websites, including numerous science-slanting websites.  The handy swipe-to-change-story feature is nice, and the entire interface is easy-to-use.  I also like using this while I'm walking because it's easy to pull up a short story that I can finish quickly and make myself feel like I accomplished something.

QuickOffice:  I'm not a big fan of paid applications - I am a poor graduate student after all. QuickOffice came preinstalled with my phone, and I must say that I am quite impressed with it.  It has the same features as the Microsoft Office suite (I can even view my PowerPoint slides on my phone!) and is really easy to use.  If you're not up for shelling out $15 (!), you can always opt for the Google Docs app, which I think is almost just as good, and you can't beat the price...

Google Reader: I use Google Reader religiously for keeping up on journal articles.  This handy little app presents my RSS feed conveniently and in my pocket so that I can keep up on my papers that I need to read.  Since I can't access the actual papers on my phone, I star the articles of interest and download on my computer when I'm connected to my university's network.

The Weather Channel:  I work in a lab with no windows.  Most of the time, I can't tell is there's a tornado outside or a beautiful sunny day.  Sometimes, that's for the best because then I'm not tempted to leave my benchtop and wander outside.  When I do have to go outside, however, it's nice to know the weather, and the Weather Channel app is convenient for quickly checking the weather.  Tons of functionality are included, such as animated weather maps.  There are a lot of weather applications out there, but I find this to be the most useful.

WTFSIMFD:  Hands down, my favorite food app.  WTFSIMFD provides you with a recipes that you should eat.  The app has a potty mouth, but it's amusing and endearing at the same time.  It also gives great recipes, you know, for when you're not in the lab or studying...

Just a roundup of what I find useful as a student and a scientist.  Maybe a followup post will be necessary when I find even more useful apps...

Mendelian Genetics Part I

Gregor Mendel was an accomplished scientist, though he never would know this during his time. His studies of pea plants (as well as other organisms) laid the groundwork for the genetic breakthroughs that would come after his death. Mendel was a Augustinian monk that had ample time to experiment with his pea plants. The principles he was able to extract from his studies are the basics taught in high schools around the world.

 Mendel studied peas, but not just any peas. His peas were true-breeding, meaning that they self-fertilized and produced essentially clones of themselves.  Over the years of study, he bred plants with specific characteristics and then crossed different varieties of pea plants to test what their offspring would look like.

Importantly, Mendel considered discontinuous, contrasting traits: he only considered the traits that he could observe (such as color), came in a limited number of forms (green or yellow) and were easily distinguishable.  For these monohybrid crosses, Mendel considered seven different traits: seed shape (round versus wrinkled), seed color (yellow versus green), pod shape (full versus constricted), pod color (yellow versus green), flower color (violet versus white), flower position (axial versus terminal), and stem height (tall versus short).  Also important to Mendel's work was his use of mathematics and statistics to estimate the probabilities of a certain type of plant emerging from a certain type of pea plant cross.

The monohybrid cross is the consideration of one particular trait at a time.  For example, if looking at pea pod color, one might cross a yellow and a green pea plant and then determine how many of the offspring had yellow or green pea pods.  This type of cross is illustrated to the right in what's called a Punnett Square.  A Punnett square is a way of organizing the different traits expressed by the individuals being crossed.  The capitalized letters are the dominant traits, while the lower-cased letters are recessive.  Vertical columns traditionally represent females; horizontal, males.  Using the Punnett square, we can look at all the possible offspring that can emerge from a cross, which can then be used to determine probabilities associated with the offspring's traits.

From these monohybrid crosses, Mendel was able to make a few conclusions.  When he crossed two pea plants of distinct traits (the P1 generation, true-breeding variety) to produce progeny (the F1 generation) and then used the progeny to generate more progeny (the F2 generation), he found that the two parental traits were still present and unchanged.  This led to the hypothesis that each parent contributed equally to the inheritance of the "genetic determinants," which would have more technical and molecular definitions in the distant future.  These determinants were separated and segregated randomly to make gametes and produce the next generation of pea plants.

Mendel's principles from the monohybrid crosses can be summarized as follows:

1. Hereditary determinants (unit factors) control traits that are in pairs in an individual.
2. When two dissimilar unit factors for a trait are combined in one organism, one factor is dominant to the other recessive factor.
3. When gametes are formed, the pair of unit factors separate and are equally likely to be separated into a gamete: the principle of segregation

In the next post, we'll examine dihybrid crosses and some more of the interesting findings from, of all things, pea plants.

MAP Kinase Signaling

Cells exist in a very dynamic environment, not only on the inside of the cell membrane, but also on the outside.  Thus, cells must be able to interact with their outside environment and respond to stimuli appropriately.  One of the several signaling pathways involved in communication from the outside of the cell inward is the (very general) MAP kinase (MAPK) signaling pathway.  The cell uses the MAPK signaling pathway for several reasons, primarily to amplify signals.  Additionally, aberrant MAPK signaling is implicated in several types of cancers.

First, MAPK signifies mitogen activated protein kinase - a fancy word for a protein that responds to mitogens, or a molecule that stimulates mitosis.  Proteins of the MAPK family were discovered in 1989 in yeast, with ERK1 being the first mammalian MAPK signaling protein, involved in insulin signaling pathways (at the time at least...).

The MAPK signaling pathway is often called a cascade because the components in the pathway amplify a signal within the cell.  The three major components are:

• MAPKKK / MEKK / MAP3K: The MAP kinase kinase kinase.  This protein phosphorylates MAP kinase kinase.  Several forms are found within the cell and their abundance is lower than that of MAPKK or MAPK; thus, MAPKKK isn't as involved in the amplification of signals.

• MAPKK / MEK / MAP2K: The MAP kinase kinase.  MAPKK phosphorylates MAPK and is highly abundant within the cell, but its only substrate is MAPK.  Just a few phosphorylation events on MAPKK result in significant activation of MAPK.

• MAPK / ERK: The MAP kinase.  MAPK is also abundant in the cell and has diverse substrates, including itself.

There are several pathways that involve MAPK signaling.  Two of the important ones are:

1. The ERK pathway: growth factor stimulation of cell surface receptors (via receptor tyrosine kinases [RTKs]) causes activation of Ras, which activates Raf (MAPKKK) to activate MEK (MAPKK) and then ERK (MAPK).  ERK's several targets include Elk and Ets, which control cellular proliferation.
2. The JNK pathway: Stress on the cell results in activation of several proteins, including Rho, which goes on to activate MEKK, then MEK, and JNK.  JNK's major targets include c-Jun, ATF2, and Elk1, which control proliferation and apoptosis.

One important consideration is the specificity of MAPK signaling.  Specificity can be achieved in several manners: protein components can only "fit" other specific protein components.  Additionally, the spacial organization of a cell can affect specificity, and this type of specificity is often controlled by scaffold proteins.  

MAPK signaling is essential for many cellular processes, including proliferation, apoptosis, embryonic development, and cancer progression.  Many intricacies of the pathways are still being worked out and will provide significant insight in the future.