CHAPTER XX: Genetic ControlA) Background Theory

1. Remember that in all organisms, the genetic code in EVERY cell is identical to all the others.2. This means that chromosome #1 in one of your skin cells is the same as chromosome #1 in your

liver cells, and every other gazillion trillion cells in your body.3. So what’s the deal? How come different types of cells are so different.4. The answer lies in which genes are turned on or off, at any given time.5. Stomach cells can make stomach acid and enzymes because those genes are triggered by

hormones and turned ON in stomach cells.6. Other types of cells do not make receptors for those hormones and molecules, so those genes

are never turned on at all.7. So basically, the pattern of which genes turn ‘on’ or ‘off’ determines the fate of individual cells.8. This prevents you from growing an arm out of your face…you freak.9. Genetic control is complex in pretty much all organisms.10. Almost no traits are controlled by a single gene. Most traits and physiological responses are the

result of several (or hundreds) of genes working together and giving feedback.11. There are several ways that genes (and the protein they make) are regulated.12. Some genes are controlled by limiting the amount of mRNA available for transcription.13. Other genes are controlled by regulating the rate of translation.14. Still other genes make protein products that can be activated or de-activated.15. Since it is hard to make too many other generalizations, particularly between prokaryotes and

eukaryotes (since they regulate genes differently), we’ll begin with how bacteria control genes.

B) Prokaryote Gene Control1. Bacteria have relatively small genomes (usually only around 2000 genes) and are simple in

comparison to eukaryotes.2. There are basically two systems of genes in bacteria, which are constitutive and regulated.3. Constitutive genes are on all the time, because they are essential for life.4. An example of a protein coded by a constitutive gene would be cytochrome C, which carries

oxygen. Living cells must always have it available.5. Regulated genes are turned on and off, according to need.6. Most genes involved with metabolism are regulated genes, since food is not always present.7. Many such genes are individual, but others occur in clusters of related genes called operons.8. An operon is a cluster of gene that turns on when the proteins coded by several genes are

needed for a related function.9. The operon system was deduced through the study of the Lac Operon and the Trp operon in

E. Coli bacteria. The Lac Operon controls lactose metabolism, while the Trp operon controls the production of the amino acid tryptophan.

10. An operon works almost like a series of switches, with several different parts.11. Starting at the beginning, the first stretch of DNA ahead of the gene is called the promoter.12. The promoter is a stretch of DNA that tells RNA polymerase “HEY!! Here is the start of the

gene! Get your butt over here and transcribe me!” It’s like runway lights at an airport.13. There is a stretch of DNA directly in front of the promoter called the operator.14. A protein called the repressor recognizes the sequence of the operator and binds to the

sequence, when the gene needs to be turned off.

15. What happens, effectively, is the repressor protein parks its butt in front of the RNA polymerase and won’t move. Therefore, transcription won’t start.

16. Only when the repressor protein is moved by some trigger, will it get out of the way and let transcription happen. Until then, no mRNA can be made, and therefore, no protein.

17. Let’s take a look at how the two operon systems first discovered in E. coli work.18. The table and diagram below explain the components of the lac operon.19.

20. The Lac Operon is what is called and induced operon. Unless a trigger molecule (lactose) is present, it remains turned off.

21. It makes good sense as to why this is so. If there was no lactose available for consumption, the cell would just be wasting energy making a useless protein that did nothing.

22. Most of the time, a bacteria will also use OTHER sugars besides lactose FIRST if given a choice, because lactose is fairly difficult to break down and provides less energy than others.

23. So, lactose is pretty much a last resort fuel.24. The lac operon, like many operons is actually a component of a larger circuit of genes called a

regulon. Regulons are clusters of operons that contain genes with common functions.25. For instance, the lac operon is one of several operons that control sugar metabolism.26. Each operon turns on in a hierarchy, according to what sort of food is available.27. So how does the bacteria regulate the order that they turn on?28. There is a second protein called CAP protein, which must be present and binding to the

promoter site, or otherwise the RNA polymerase can’t bind.29. The CAP protein holds on to RNA polymerase the way a saddle lets someone stay on a horse.

Without it, transcription goes nowhere, because they enzyme falls off.30. Most of the time, there is no CAP protein bound to any operon, other than the particular sugar

that is being consumed for energy.31. When that sugar goes away, the cell sends out a cAMP signal saying “Hey I’m hungry! What else

is there to eat around here?”32. In E. Coli, the 33. The cAMP then binds inactive CAP protein, changing it to its active conformation.34. The active CAP protein then binds to the promoter, allowing the RNA polymerase to stick and

RNA transcription to go forward.35. From there, the metabolic enzymes for the sugar’s metabolism are made.36. A diagram of the entire thing is shown below.

37. So how did anyone ever figure this whole thing out anyway?38. A couple of researchers named Francois Jacob and Jacques Monod mapped everything out by

back-tracking the map layout from studying mutant strains of E. Coli. 39. First, they X-rayed a ton of E. Coli cultures. This randomly created mutations. Some of these

cultures produced mutants specific to the genes for lactose metabolism.40. From there, they found four general types of mutants, and were able to back-track and figure

out the function of the mutated proteins.41. The first two types of mutants were repressor mutants. 42. The first type of repressor mutant could not use lactose at all, because a mutation to the

repressor protein caused it to bind so tightly that it would not let go.43. As you might expect, the second type of repressor mutant did not bind at all, so the bacteria

made lactase enzymes all the time, in spite of the fact that they weren’t needed.44. Operator mutants had a faulty operator sequence that the repressor protein did not recognize,

so these mutants also produced lactase constantly.

45. Finally, they found metabolic mutants, which had problems with Lac Y, Lac Z, or Lac A. They were only able to partially metabolize the glucose.

46. On the surface (with modern technology), the premise doesn’t sound that hard, but for the 1960’s it was revolutionary. They both received the Nobel Prize.

47. Here are these two cat daddies in their lab and a schematic of the interaction of glucose and lactose when E. coli encounters various concentrations.

48. In addition to inducible operons, some genes are controlled by repressible operons.49. Repressible operons are always ON unless something stops them.50. An example of this is the Trp operon. 51. Bacterial cells will always make the amino acid tryptophan at low levels, since it is essential for

building proteins, as long as it remains unavailable.52. However, should tryptophan become available, a system is needed to shut the genes off.53. The Trp operon works in reverse of the way the Lac Operon works.54. As long as tryptophan is unavailable, the repressor protein is unable to bind to the operator,

because it is in the wrong conformational shape. 55. In this case, the genes for making tryptophan remain on, and the cell cranks out the metabolic

enzymes needed to build it. 56. If the bacteria happens to find itself on easy street, hanging out on a spoiled bratwurst or taking

a trip to the Browns game in the Superbowl, then tryptophan is suddenly available.57. Tryptophan, itself, binds to an allosteric site on the repressor protein, activating it.58. The repressor then binds the operator, until all of the tryptophan has been metabolized.59. Once all the tryptophan is gone, the repressor changes shape again, falls off, and the operon

starts up all over again.60. A picture of the Trp operon is shown below.

61. Like the sugar metabolism system, a lot of the pathways that manufacture non-essential amino acids are also controlled by regulons.

62. Regulons also control the metabolic reactions involving nitrogen compounds and phosphates.63. While the controls we have discussed so far are pre-transcriptional, there are also post-

transcriptional and post-translational controls that involve mRNA and proteins.64. For instance, mRNA can be translated into proteins at different speeds, according to how many

ribosomes transcribe the sequence and how tightly the mRNA can bind.65. In other cases, such as the repressible and inducible proteins we just talked about, some

proteins are made in an inactive state and must be activated via an allosteric site or cleaved by enzymes to become active.

C) Eukaryotic Gene Control1. Eukaryotes are extremely different from prokaryotes, genetically speaking.2. In addition to having much more DNA divided across many chromosomes, along with large areas

of introns, eukaryotes usually regulate their genes differently.3. While there are a few clusters of genes on operon systems, most eukaryote genes are not.4. Most eukaryotic genes fit into one of three categories.5. Temporal genes are usually involved with development. They only turn on once or a few times,

after which, they are completely inactivated.6. For instance, the genes that control development of your eyes, arms, and legs are temporal.7. If you lose your hand, you can’t just sprout another one, because the genes are turned off.8. Usually, enzymes called methylases do the turning off. After some developmental cue triggers

them, they add methyl groups to the DNA bases after the gene has served its purpose.9. This causes the cell to ignore the gene thereafter, since the methyl groups inhibit RNA

polymerase from accessing the DNA.10. So, a number of your genes are permanently off. You can’t start them back up again.11. However, some animals do not turn off their developmental genes.12. This explains why a lizard can grow a new tail, but you can’t grow a new butt.13. Here are pictures related to this notion. Sir Mix-A-Lot’s DNA is methylated and he cannot

regrow a butt, should he ever lose his ‘back’, while the skink is just fine and always has back.14. Sir Mix A Lot likes genetics and he can’t deny. Those other brother can’t deny, that when a

scientist walks in with an itty bitty gel box and a DNA sequencer in his face, he gets sprung.

15. A number of other genes are constitutively regulated genes.16. This means that they turn on or off as needed.17. For instance, if you eat a meal, the gene that makes insulin is turned on by the presence of sugar

in the blood. However, when you are not eating, the gene shuts off to avoid waste.18. Many of these genes are specific to tissues. For instance, the gene for insulin production NEVER

turns on in muscle cells or skin cells, because they lack the receptors to trigger this.

19. As you remember, hormones and signal molecules are used to turn on genes this way.20. Protein hormones usually bind to receptor molecules on the cell’s surface.21. Hormone proteins, on the other hand, diffuse right across the membrane and directly interact

with transcription factor proteins that control RNA transcription.22. If you aren’t clear on this concept, you might want to go back and review the chapter on the

endocrine system. There are also refresher diagrams below.23. Some genes, as they are in prokaryotes, are also constitutive in eukaryotes, particularly the

ones under the control of mitochondria or chloroplasts.24. It’s not like you can ever afford to stop making cytochrome C or hemoglobin, for instance. That

is…unless you don’t value not suffocating to death.25. Finally, other genes are for emergency only, and are triggered by such conditions.26. Most of these genes produce chaperone proteins, heat shock proteins, or drought shock

proteins. These proteins are made to prevent other proteins from becoming denatured.27. Chaperone proteins literally push back on proteins that are starting to denature and keep them

in their correct shape.28. The three pictures below show the different scenarios with the different types of genes.29. Development is temporal. Digesting food involves constitutive genes and avoiding death by

enzyme denaturation in the heat of the day involves emergency genes in the cactus.

30. As mentioned earlier, very few of these genes are under the control of operon systems.31. Most eukaryotic genes are individually controlled, though they interact with other genes and

cause their transcription to be started, stopped, slowed, or speeded up.32. To better understand the process, we will break down the regulation methods into pre-

transcriptional (before mRNA is made) and post-transcriptional/post-translational (after mRNA or protein products have been made).

D) Pre-Transcriptional Control of Eukaryotic Genes1. Transcription is the level where most eukaryotic genes are regulated.2. Common sense says that if you don’t need a gene product, its best not to bother making it at all.3. While the mechanisms are different than they are in prokaryotes, eukaryote genes also have

systems that allow them to be switched on and off.4. To understand the regulation of eukaryotic genes, we will break down the parts of a typical

gene sequence. The box below describes each component and the schematic shows it.

As

Component Function

Promoter Region recognized by RNA polymerase that notes the start of a gene. Usually around 30 to 50 DNA bases long.

Promoter elements Most promoter sequences have regions of repeated DNA sequences in their content that bind the RNA polymerase enzyme. The most common of these are the TATA box and the CAT box, which repeat over and over.

RNA Polymerase The enzyme that makes a complementary mRNA copy to the DNA gene.

Start Codon The start codon of any eukaryote gene is always AUG, which codes for the amino acid methionine. This can later be processed off if needed.

Gene Codons (Exons) This is the main segment of DNA that codes for the protein. This segment can be anywhere from a few dozen to hundreds of thousands of codons long.

Introns Many genes are interrupted with junk sequences. However, these get transcribed right along with the exons, and they must be processed out later.

Stop Codon The sequences UGA, UAA, or UAG all tell the RNA polymerase that its at the end of the line and its time to fall off. The end of the gene.

Upstream Promoter Elements

These are sequences of DNA usually several hundred DNA bases upstream of the promoter. The more of these that are present, the stronger the RNA polymerase binds, and the faster the gene is transcribed.

Enhancer Sequences These sequences are found fairly far away upstream from the promoter region of the gene. These sequences bind transcription factor proteins.

Transcription Factor Proteins

There are many types of these. These proteins bind enhancer sequences and then bend until they find an RNA polymerase enzyme. These regulate the speed of transcription. Generally, the more that are bound, the faster the transcription goes, and the quicker the protein is made.

Repressors If transcription factors are the gas, then these are the brakes. These slow down transcription when they bind. Sometimes these are proteins, but sometimes they can be other chemical signals.

Proximal Region This is a region of DNA this is fairly close to the promoter. This is where steroid hormones bind and interact with transcription factors and RNA polymerase.

Steroid Hormones Steroids bind at the proximal region and speed up or slow down the speed of transcription, depending on what gene they interact with and the type of hormone. They do this by pushing or pulling on transcription factors and RNA polymerase.

5. As the table above outlines, there are several different types of proteins that interact with varying speeds and intensities to control transcription.

6. This explains why certain genes are expressed in some cells and not in others. 7. Now let’s turn to how the proteins, themselves, grip the DNA and how they work.8. Transcription factors and repressors, as well as the active domains of polymerase enzymes

usually contain domains (regions of the protein) that have common design functions.9. There are three general motifs used for DNA-binding proteins.10. Helix-turn-helix protein domains have two facing regions of alpha-helices in their secondary

structure. The amino acids that sit in these ridges have R-groups that interact with the side chains of DNA bases, causing them to stick together with hydrogen bonds.

11. Zinc-finger protein domains have several small stretches of amino acids called fingers. Each finger has R-groups that hydrogen bond to specific stretches of DNA. The fingers are all held together by a zinc ion, which chelates them.

12. Leucine zipper protein domains occur as dimers (two parts) and are shaped like a Y.13. They are called leucine zippers, because long stretches of repeating leucines interact with one

another and hold the Y together into shape.14. The forks of the Y contain amino acids whose R-groups bind the DNA.15. Pictures of the three types of DNA-binding protein domains are shown below.

16. Some proteins are needed in such great quantities (hemoglobin and other oxygen-carrying globulin proteins for instance), that transcription going really fast is not fast enough.

17. There are certain genes that may be present in multiple copies. Therefore, the same gene is being transcribed several times at once. This is called gene amplification.

18. Genes that are extremely active are located in areas of the chromosome called euchromatin.19. Euchromatin is very loosely packed, as compared to the rest of the chromosome, so

transcription factors and enzymes can easily access it.20. In contrast, some DNA is located in densely packed areas called heterochromatin. 21. Most of the genes in heterochromatin are either rarely active, or more commonly, permanently

inactive developmental genes, which are methylated to preven them being turned on again.22. In females, almost an entire X-chromosome becomes methylated and inactivated.

Leucine Zipper Protein MotifHelix-turn-helix protein motif Zinc finger protein motif

23. This extra chromosome is called a Barr body and is very conspicuous under a microscope. It shows up well, because it’s so densely packed, it takes up lots of stain.

E) Post-Transcriptional and Post-Translational Genetic Regulation in Eukaryotes1. As previously mentioned, most of the time, it is more efficient for a cell to pre-emptively control

protein production before a gene is ever transcribed into mRNA.2. However, there is a need for processing after transcription for other reasons.3. In most genes, introns are not edited out during transcription.4. This means that left unattended, most proteins would have large areas of non-functional junk in

them that would ruin the protein.5. Therefore, the cell must process this junk out.6. Enzymes called sNRPs (small nuclear ribonuclear proteins) are complex enzymatic proteins that

are in charge of chopping out the superfluous mRNA and piecing it back together.7. So the sNRPs cut out all of the junk mRNA and give the ribosome a usable transcript.8. In this case, RNA polymerase is like a long-winded newspaper columnist like Dear Abby that just

won’t shut up. She sends in a transcript to be printed in the paper. 9. The sNRPs are like the editors who cut her crappy column down to a paragraph.10. The ribosome, then, is analogous to the printing press that sends out finished papers (proteins).11. Additionally, some proteins are modular, with a quaternary structure that must be put together

from multiple puzzle pieces.12. Again, the sNRPs step in and cut out the modules and glue them together, as if they were

assembling cheap cardboard furniture from Wal-Mart, except understanding the complexities of molecular biology is easier than assembling do-it-yourself furniture made in Hong Kong.

13. Sometimes, this means that the plans of the protein come from the mRNA from separate genes.14. Other times, it means that different patterns of gene splicing can result in multiple combinations

of mRNAs that give you multiple different proteins.15. An example of a real life protein that is spliced differently in different tissues is troponin.16. You may remember that troponin is the protein in muscle fibers that acts like a lock on a bicycle

rack. Before you can contract a muscle, troponin must be removed.17. Troponin happens to have slightly different structures in skeletal, cardiac, and smooth muscle.18. However, there is only ONE gene for troponin in all cells. What makes it different in different

cells is the way that the mRNA is spliced up and glued back together by sNRPs.19. sNRPs are also responsible for helping to advance the philosophies and ideologies of

communism, behind their red-hatted leader Papa sNRP. It is a mystery how sNRPs replicate themselves, since sNRPette seems to be the only female sNRP in the village.

20. La-La…La-La-La-La….sing a happy song, Friedrich Engels.21. By the way, Gargamel represents capitalism on the Smurfs, which is why he is depicted as evil. Is

it really a coincidence that he wanted to turn the smurfs into gold?22. The snRPs also lived in a classless society, a la Karl Marx’s communist manifesto.23. The diagram below simplifies the differential splicing idea.24. Eventually, the mRNA makes its way over to the ribosome, where it is translated into protein.25. However, mRNA can’t just be left lying around, or it will continue to go back through the loop

over and over again, and tons of waste proteins will be made.26. For this reason, the cytoplasm is full of recycling enzymes called RNAses that chew up the RNA

and recycle it into A, C, G, and U.27. There is, however, a happy medium between immediately destroying the new RNA with RNAses

and leaving it lying around forever.

28. Most eukaryote mRNAs are fairly long-lived, lasting for the better part of a day. The secret is in the poly-A tail. At the end of transcription in eukaryotes, RNA polymerase will add a long stretch of meaningless adenines.

29. This sequence is ignored by the ribosome, since it comes after the stop codon.30. However, this gives the angry RNAses something to chew on, before they finally start chomping

away at the useful part of the transcript. The poly-A tail works something like a timed fuse.31. In some cases, RNAses are also inhibited by certain hormones.32. For instance, vitellogenin is a protein that packs eggs full of stored proteins like albumen.33. If a female animal is ovulating, this means that there are high estrogen levels.34. In turn, the hormone estrogen triggers genes that cause RNAse enzymes to become inhibited

and slow down. This allows the RNA for vitellogenin protein to stick around, until the egg is full of scrumptious protein goodness.

35. In addition to post-transcriptional processing, there are also some proteins which do not become active until post-translational processing.

36. For instance, some proteins are made with included intron sequences that have been translated into protein.

37. Enzymes in the golgi apparatus must splice out these junk segments, in order for the protein to become active and useful.

38. Additionally, some proteins must be spliced in other ways, combined with other molecules, or assembled into their quaternary structure from multiple pieces to become active.

39. An example of this would be the cyclin proteins used to control the cell cycle. These must be phosphorylated by kinase enzymes, in order to be activated.

40. This is because the cell doesn’t want the protein doing its thing until it’s ready to divide. If the cyclin proteins went around telling the cell to divide constantly, you would grow a tumor.

41. Insulin is an example of protein that must be activated by other enzymes. In its case, a piece of the protein must be cut off, in order for it to activate.