"It was the best of times, it was the worst of times." No you aren't in English class and no we are not about to ask you about allegories, epigraphs, or allusions. What we will say though, is that maybe A Tale of Two Cities isn't too irrelevant to biology. It turns out that change and suffering isn't limited to the 18th century France. Imagine that.
Organisms, and the cells that comprise them, are constantly exposed to different conditions, good and bad. Adaption is critical for survival. Now if you are thinking of all-nighter video game marathons, rock concerts, sunbathing, and trips to Antarctica, and you are totally on track.
You can also think about a single-celled organism like yeast. A single cell sits on a grapevine, exposed to rain, wind, and drought. These conditions generate stressful situations for the cell, where food and water may not be as abundant or readily available. Extreme temperatures can also lead to non-ideal growing conditions.
Cells respond to changing conditions by changing the amount (and types) of proteins they produce. But wait, let's back up a little and talk about how the amount of a protein differs from one cell to another in the first place.
Different cells produce different amounts of proteins. Some proteins are uniquely expressed in certain cell types or organisms. Other times it is only the levels of proteins that are different. This straightforward concept best describes why each of the cells in your body is capable of playing the role it does. What we have told you is that the amount of any given protein differs from one cell to another. The amount may also be changed as the cell adapts to its environment. For example, a single celled organism exposed to high heat may start producing a type of protein that helps other proteins fold at the higher temperature.
That brings us to a basic question: how does a cell control the amount of protein it produces? You know all about transcription and translation now, so can you think of some ways that a regulatory system might work?
Not too long ago some of the world's top scientists thought it was by getting rid of DNA that the cell didn't need. Can you think of why this situation would be a bad solution for the cell? It is kind of like throwing out all of your winter clothes in summer because it is hot at the moment. Come wintertime you'd be a unhappy camper.
You've probably figured out that the cell can alter the transcription of RNAs and well as the translation of proteins to create the desired protein output. All of this work can be done without altering the amount or quantity of DNA in the cell. Without further ado, let's start talking about how transcription and translation can be modified to create different amounts of proteins.
The cell can regulate the amount of protein produced by regulating the amount of mRNA; this is akin to killing the messenger. This way the cell doesn't need to make a whole bunch of RNA that it won't end up using. This is the method that is most commonly used to regulate the production of a protein.
One of the most common ways to control transcription is through the use of specific DNA binding factors. These proteins can recognize specific DNA sequences around the transcription start site. They can provide all sorts of levels of control, from a simple on and off switch, to more complex regulatory control. Think of a light on a dimming switch.
Let's start off with a simple switch first from…you guessed it…bacteria! Those little suckers are great at providing straightforward example of how transcription can be regulated. Sensing a pattern yet? Good. It is a general rule of thumb that eukaryotic processes are lot more complicated than prokaryotes. It is for this reason scientists like to study prokaryotic processes first. It is kind of like learning to ski on the bunny hills before jumping to Corbet. Insert link http://www.travelandleisure.com/articles/worlds-scariest-ski-slopes
What does gene control look like in prokaryotes like bacteria?
A lot of bacterial genes are arranged in operons, groupings of similar genes arranged in a similar location in the genome. These genes can be controlled all at once and even used to produce a single mRNA. Most often these genes are all involved in a particular process in the cell. Operons are often controlled by the regulatory sequences located adjacent to the gene. This type of regulation is called regulation in cis.
Psst…don't confuse operon with Oberon, the king of the fairies. You might remember that dude from Shakespeare's A Midsummer Night's Dream. He's the one that makes Titania fall in love with Bottom, a weaver wearing a donkey's head. I guess now that we think about it, operons and Oberon do have something in common. They are both all about control, but operons don't work though love potions. Thank goodness, too, because biology is complicated enough as it is without getting Cupid involved!
Alright, back to biology. Oftentimes RNA polymerase can't begin transcription without the assistance of regulatory DNA binding proteins. These proteins bind to the promoter in a special region called an operator. Since these binding proteins activate transcription through the RNA polymerase, they are called positive regulators, working through positive regulation. Alternatively, a different set of proteins, called transcriptional repressors, can bind to the genes preventing their transcription. This is a form of repression, or negative regulation. Regulation that involves the binding of either positive or negative regulatory proteins is called regulation in trans because they involve (trans) factors separate from the DNA sequence. We'll show you a real life example in a minute.
To keep cis and trans regulation straight, it may be helpful to remember the roots for cis and trans. Cis means "on the same side", while trans means "on the opposite side" or "beyond."
Most often genes are controlled by a combination of both positive and negative regulation. One of the most commonly described example in prokaryotes is the lac operon in E. coli. Genes in the lac operon are important for the transport and catabolism of the sugar lactose. Lactose is used as a sugar source only when the sugar glucose is absent or low. The operon is controlled such that the genes are only produced when lactose is present and glucose is absent. Both conditions must be met. Having lactose present is not enough.
The way the lac operon works is that the cAMP activator binds to the promoter of the lac operon only when lactose is present. The repressor binds when the cell has high levels of glucose. If the repressor is present RNA polymerase cannot bind. Only when the repressor is absent will the polymerase be able to bind. The cAMP activator is then required for the polymerase to actively transcribe the genes.
In some instances genes can be regulated by another type of regulation, called feedback regulation. In this case, the protein produced by a gene itself comes back to regulate the gene, either positively or negatively. These type of regulation provides a type of loop that allows the cell to gauge how much of a product it was produced. Pretty, pretty smart.
Yeah…that was the straightforward example….
Similar but more complicated regulation examples are also observed in eukaryotes. Why does regulation need to be so much more complicated in eukaryotes like us?
One reason for this situation is that eukaryotic genes are located within the context of higher order chromatin structure. Proteins that influence the structure of the DNA surrounding a gene can influence the ability for the polymerase and related factors to bind to the promoter. In fact, eukaryotic chromatin is often described as either heterochromatin or euchromatin.
Eukaryotic DNA is more open, and contains much of the regions of the genome that are actively transcribed. This "openness" is important for allowing regulatory proteins and RNA polymerase to bind. Alternatively, heterochromatin is more densely packed, and contains regions of the genome that are not likely to be transcribed. This "closed" structure of DNA can be compared to the difficulty in reading a novel with the cover closed. The DNA sequence is not just accessible for reading. Changing the chromatin structure surrounding a gene is another way that a cell can control a eukaryotic gene's transcription.
Another key difference in eukaryotes is the regulatory sequences are often located much farther away from genes. Eukaryotic activators, often called enhancers, can be located thousands of base pairs from the promoter. Activator proteins can bind to these DNA regions and bring them closer to the vicinity of the promoter. This can influence the ability of the polymerase and transcription factors to bind. Repressor proteins can work by a similar long distance mechanism in eukaryotes. Complicated stuff. No one said being a biologist was easy.
As we mentioned earlier, it is also possible to prevent the production of a protein by repressing a step other than transcription. RNA splicing, RNA transport, RNA stability, and the progress of translation are all steps where regulation can and does occur. Let's talk about some examples of how these different steps can be regulated.
Transcription and translation overcome a number of obstacles to produce functional RNAs and proteins. You've probably guessed by now that the structures of the factors involved are absolutely essential for the success for these processes. Are you beginning to feel a little déjà vu here? That's because the link between structure and function is such an important theme in biology.
Many of the common factors involved in transcription and translation show remarkable similarity across organisms (read: similar structures used for similar functions). These factors exploit common principles to fulfill their functions. First let's talk about proteins involved in transcription.
We've talked about how the proteins involved in transcription must recognize specific regions of DNA. The promoter. The regulatory regions. Proteins don't have eyes, but they can "read" DNA in a non-traditional way. In fact, the DNA helix can be read from the outside of the helix. Why is this observation surprising?
Look at the structure of the DNA helix below. The DNA helix has both minor and major grooves, but the base pairs hydrogen bond within the center of the helix. What does this mean? Basically the factors involved in transcription are reading a book by looking at its cover.
It is actually not quite as complicated as you might think. The DNA bases do expose an edge of the base, and this fact is enough for proteins to figure out the inner sequence. It is kind of like closing your eyes and trying to pick out a particular fruit from feeling along the edges of the fruit in the bowl. It is also easier for some sequences to be recognized because they generate a distinctive kink or bend in the helix. The proteins that bind to these regions must form complementary interactions with these exposed residues.
There are certain common DNA structural binding domains, or parts of a protein important for binding DNA, which are observed in many DNA binding and regulatory proteins. In other words, these domains look alike and share common functions. Imagine that!
Some common domains are helix-turn-helix and zinc fingers. Helix-turn-helix are formed by two protein α-helixes. Different helix-turn-helix proteins have unique amino acids sequences despite sharing a common protein structure, and these differences provide the specificity to recognize the specific DNA sequence. Zinc fingers motifs use amino acids and zinc to recognize specific DNA structures. Both helix-turn-helix and zinc fingers use α-helixes to recognize the major grove of the DNA. These groups of proteins also tend to function as dimers, or a pair of two molecules. Although less common, amino acids arranged into β-sheets can also contact the DNA helix through the major groove.
The importance of structure isn't unique to proteins. We've mentioned that even the nascent RNA strand can take on unique structures to aid in its release from the polymerase. Furthermore, the RNAs involved in the production of proteins take on elaborate structures, all of which are essential for their function. Like proteins, RNAs can form elaborate structures and even catalyze biological reactions.
RNA also has the unique advantage that they can base pair with DNA and other RNA structures. We've already talked extensively about how this base pairing allows for the codon/anticodon pairing required for translation. Base pairing is also how the RNA can take on higher order structures by base pairing to bases within the same molecules. Disruption of these interactions can be extremely disruptive, even leading to a tRNA that charges with an incorrect amino acid. Sound familiar? Yes, good. You've been paying attention. If not, click here for a little refresher—link to section on tRNAs.
Up until this point we have talked about transcription and translation as though all the players magically came together. But in reality, it is a lot more complicated. DNA is in the nucleus in a eukaryotic cell. Where do you think transcription must occur? Where do you think proteins are made? What might happen if the proteins or RNA didn't make it to the correct spot?
Let's start at the beginning. DNA is in the nucleus in a eukaryotic cell. Transcription occurs in the nucleus. Therefore, all the transcription stars, such as polymerase, transcription factors, regulatory proteins, need to be there too.
Once an mRNA is made, the RNA immediately undergoes processing. Splicing and capping occur. These covalent modifications are important, because they indicate that an mRNA is complete and ready to transport. The completion of processing is the green flag for the RNA to begin its transport out of the nucleus.
You may remember that the nucleus is a membrane bound organelle. Think of this barrier as the bouncer of nucleus. Not anyone will get by this dude. In fact, the mRNA needs to have its own connection to get by him. This connection is called the nuclear pore complex.
The nuclear pore complex is pretty much what it sounds like: a pore where proteins and RNA can be released into the rest of the cell. A specific group of proteins, called poly-A binding proteins, bind to the mature mRNA and guide it to the nuclear pore complex so it can "sneak" out of the nucleus into the cytoplasm. It is here in the cytoplasm where translation takes place: on the rough endoplasmic reticulum, or on free-floating ribosomes. The proteins produced by translation then are transported to the correct regions of cell such that they can fulfill their functions.
What about the RNAs that don't have a poly-A tail and aren't mRNAs? Great question. The transport of rRNAs and tRNAs is closely linked to their transport as well. rRNA are produced and processed in a special region of the nucleus called the nucleolus. This region isn't membrane bound, but it is visible under the microscope. It contains the many copies of ribosomal RNA genes needed to produce the mass amount of rRNA needed by the cell.
What we have told you is that transcription and translation, and the components involved in both processes, are localized to specific regions of the cell. Pretty important stuff. Now though, we'll leave you with three important questions to ponder.