Control of Gene Expression in Eukaryotes

Control of Transcription

• Focuses on the control of transcription in eukaryotes, specifically whether messenger RNA is made or not.
• The control of transcription in eukaryotes is analogous to prokaryotes in that it takes place at the promoter.

Eukaryotic Gene

• Eukaryotic genes consist of double-stranded DNA, introns, and exons.
• The promoter is the most important part; it is where RNA polymerase interacts with the DNA to begin making complementary RNA.

RNA Polymerases

• Eukaryotic RNA polymerases cannot function independently, unlike prokaryotic ones.
• RNA polymerase requires assistance to begin transcribing.
• In vitro experiments show that RNA polymerase does not transcribe the gene on its own.
• Transcription factors are proteins that associate with the promoter and RNA polymerase to facilitate transcription.
• The transcription factors interact with both the RNA polymerase and the promoter, forming a complex.
• Once the complex is formed, RNA polymerase can start making RNA, moving down the gene to transcribe it.

Transcription Factors

• There are many different types of transcription factors.
• Transcription factors interact with specific promoters, and their activity can be turned on and off by the cell.
• When transcription factors are activated, they move to a specific promoter and allow RNA polymerase to make RNA.

Example: PSA Gene

• The PSA (prostate-specific antigen) gene is present in all humans, but it's primarily made in the prostate gland of biological males.
• PSA protein is only manufactured where there are appreciable amounts of testosterone, typically post-puberty in biological males.

Androgen Response Element

• The promoter of the PSA gene contains a region called the androgen response element (ARE).
• The ARE is a specific DNA sequence that signals a specific transcription factor called the androgen receptor (AR).
• The AR protein, a transcription factor, interacts with the promoter and promotes the expression of genes with this ARE.

Mechanism of Testosterone

• In biological males with no appreciable amounts of testosterone, RNA polymerase cannot transcribe the PSA gene due to the lack of a transcription factor.
• When testosterone is introduced into cells, it interacts with the AR protein.
• Testosterone, as a steroid, can diffuse through the phospholipid bilayer and enter cells.
• The AR protein interacts with testosterone, causing a change in its shape and function.
• The activated AR protein moves to the androgen response element, facilitating the interaction between RNA polymerase and the promoter.
• The complex then starts moving down the gene, transcribing the messenger RNA for the PSA protein.
• Transcription factors often work in pairs when in their active form to form the active complex; such as the Janus transcription factor.

Enhancers and Activators

• Activators work similarly to transcription factors but bind to regions called enhancers instead of the promoter.
• Enhancers can exist before or after the gene
• Once activated activators bind to enhancers.
• Activators and transcription factors find each other causing the complex to fold and loop.
• This complex then promotes the activity of the RNA polymerase.
• Like transcription factors, activators interact only with specific enhancers and can be turned on or off by the cell.

Post-Transcriptional Control

• Involves modifying the RNA after it has been made.

Alternative Splicing

• Different introns and exons can be removed, resulting in different messenger RNAs.
• This can lead to the production of different proteins with different activities.
• Different transcripts are seen for most genes.

RNA Interference

• Involves using microRNAs to inhibit the translation of messenger RNA.
• If a cell no longer wants to make a certain protein, it makes a microRNA complementary to a region of the messenger RNA.
• The microRNA and messenger RNA form a short region of double-stranded RNA, which cells recognize and destroy using a protein complex called the dicer.

Post-Translational Modification

• Involves modifying the protein after it has been made.

Modification of the Protein

• Attaching things such as carbohydrates to it.

Ubiquinization

• A mechanism where the cell destroys individual proteins.
• The cell attaches ubiquinol proteins to the protein marked for destruction.
• A protease specifically recognizes these ubiquinol proteins and hydrolyzes the protein into its constituent amino acids.

Polymerase Chain Reaction (PCR)

• The polymerase chain reaction makes countless copies of one small area of the DNA of the genome.

Required components:

• Template DNA (the original DNA to be copied).
• DNA polymerase (an enzyme that copies DNA).
• Nucleotides (the raw material for the DNA polymerase).
• Single-stranded DNA primers (complementary to the region of interest).

Primers

• The primers are single-stranded DNA, and they're going to act as primers for the DNA polymerase
• Since DNA polymerases need preexisting nucleic acids to attach the brand new nucleotides, the DNA primers are analogous to the RNA primers in DNA replication.
• The reason this works is because all the regions of the DNA, if you make the primers properly, they are complementary to only this region of all of the gigabytes of information in your genome.

DNA Extraction

• Typically, scientists would concentrate and purify the DNA, but for the experiment being done in a lab, the crude lysate is fine for use.

PCR Tubes

• PCR occurs in tiny tubes called PCR tubes that fit in an instrument called the thermocycler.
• Inside the tubes, that contains a bead and that bead has most of the stuff that the PCR needs: DNA polymerase, nucleotides, and buffers.
• PCR primers (single stranded DNA fragments complementary to the region)

Thermocycler

• The thermocycler is a device that quickly heats and cools down this metal block, very rapidly heats and cools it down.
• So the solution on the inside heats and cools down quickly as well. It's about this big, and the metal block is about this big. We can fit 96 of these PCR tubes in the metal block at once.

Steps of PCR

1. Denaturization

• The PCR machine rapidly raises the temperature up to nearly boiling (95°C).
• This unzips the DNA, separating the two strands.

2. Annealing

• The temperature is lowered (typically to 55-65°C).
• This allows the PCR primers to find their complementary regions and anneal to the single-stranded DNA.

3. Elongation

• The temperature is raised to the ideal temperature for the DNA polymerase (73°C).
• The DNA polymerase uses the primers as a starting point to add new nucleotides and copy the DNA.

Multiple Cycles

• The thermocycler repeats this process, doubling the amount of target DNA with each cycle.
• In lab, the program goes through this 25 times, but depending on what is being done, you can go up to 40 times.

Taq Polymerase

• In Yellowstone National Park, there are bacteria called thermos aquaticus that have evolved to live in warm temperatures.
• Their DNA polymerase, called Taq polymerase, is used for PCR because it can withstand the high temperatures required for the process.

Application of PCR

• Researchers are phenotyping students again for the ability to taste the bitter PTC compound, and they already know what mutation causes this.

Tasting ability

• The sequence of g g c g c c a c t codes for the amino acid proline.
• A third of the people have this switch to c, which makes the amino acid alanine.
• The dominant allele has proline so they can taste, and they can't taste the recessive one.

Restriction Enzyme

• The restriction enzyme cuts the DNA sequence of g g c c, but it is sensitive to the shape of the molecule.

Gel Electrophoresis

• Gel electrophoresis will be run through a viscous medium, and the speed at which the DNA runs depends on the size.

Agarose Gel

• Agarose is a seaweed carbohydrate, and the gel will be in a box filled of salt water (buffer) to conduct electricity.
• The DNA will be put into the wells, and then the electric voltage is put on.
• The DNA is negatively charged because of the phosphate groups, so the DNA will go towards the positive terminal.
• The smaller the strand, the faster it goes through the gel.
• Fragment has 202 base pairs but one is 80 and one is 20. With one the 81 and the other being 20.