RR2: Eukaryotic Transcription

Question 1

What mostly controls gene expression?

Answer 1

73%: rates of transcription.
11%: rates of mRNA degradation
8%: rates of protein degradation
8%: rates of mRNA translation

Question 2

Do an overview of transcription.

Answer 2

1. RNA polymerase recognizes dsDNA.
2. DNA double helix locally denatures.
3. One strand acts as a template.
4. Incoming rNTPs base-pair with bases on the template strand.
5. RNA polymerase joins the rNTPs from 5’ to 3’on the new RNA.

DNA is read from 3’ to 5’

Question 3

Why is polymerization energetically favoured in transcription?

Answer 3

Because the high energy bond between alpha and beta phosphates on the rNTPs are replaced by a lower-energy phosphodiester bond.

Question 4

Where does transcription start?

Answer 4

At +1.

Question 5

Is the promoter upstream or downstream of the start site?

Answer 5

It can be either upstream (negative numbers) or downstream (positive numbers)

Question 6

Is transcription going upstream or downstream?

Answer 6

It’s going downstream of the start sign (in the positive)

Question 7

What’s a coding sequence?

Answer 7

A region of DNA or RNA whose sequence determines the sequence of amino acids in a protein.

Question 8

At what rate does RNA Pol 2 go?

Answer 8

1000 to 3000 nucleotides per minute. which is pretty fast but not super fast

Question 9

What’s the longest human gene and how long does it take to transcribe it once?

Answer 9

Dystrophin (DMD) and it takes a day to transcribe.

Question 10

What are the 3 stages of transcription?

Answer 10

Initiation
Elongation
Termination

Question 11

What’s the first stage of the 3 transcription stages and explain.

Answer 11

Initiation
Polymerase binds to the promoter sequence,
locally denatures the DNA,
catalyzes the first phosphodiester linkage.

Question 12

What’s the second stage of the 3 transcription stages and explain.

Answer 12

Elongation
Polymerase advances 3’ to 5’ down the template strand,
denaturing the DNA and
polymerizing the RNA

Question 13

What’s the last stage of the 3 transcription stages and explain.

Answer 13

Termination
Polymerase recognizes a stop site, releases the completed RNA and dissociates from DNA.

Question 14

What are the 3 aspects that are the same in both prokaryotic and eukaryotic transcription?

Answer 14

𝜎 Factors
- 𝜎 Factors confer specificity to RNA polymerase (so it binds to a specific sequence that needs to be transcribed)
regulation of the rate of RNA synthesis
- DNA binding proteins regulate the rate of RNA synthesis by enhancing or impeding RNA polymerase binding to promoter regions
DNA proximal
- Sequences in the DNA proximal to the transcribed gene are critical for efficient transcription

Question 15

lacZ is a reporter gene, what would need to bind to the promoter to stop lacZ from being trasncribed?

Answer 15

A lac repressor.

Question 16

What if we wanted to increase the rate of transcription, what would need to happen?

Answer 16

CAP would bind to the CAP site, and then cAMP would bind to CAP.

Question 17

What are some differences between prokaryotic and eukaryotic transcription?

Answer 17

Prokaryotic transcription has multiple transcription start site, while eukaryotic has only one.
Prokaryotic transcription changes very rapidly because environments control what genes have to be expressed. Those fast changes require allosteric regulation, while eukaryotic transcription isn’t changing rapidly because of the environment and doesn’t use as much allosteric regulation.
Prokaryotes also use polycistronic mechanisms to activate fast transcription. From one mRNA, you can get multiple different proteins.

Eukaryotes are monocistronic.
In Eukaryotes, transcription is used to differentiate cell types. The changes in transcription efficiency and the activation of specific genes that allow us to distinguish the various cells that make up an embryo.
Eukaryotes have 3 types of RNA polymerases.

Question 18

What do polycistronic and monocistronic mean?

Answer 18

Polycistronic: Describes an mRNA corresponding to multiple genes, so this mRNA can give rise to multiple different proteins.

Monocistronic: Describes an mRNA that has one cistron in a transcription unit, so only one structural gene that codes for one protein.

Question 19

What is allosteric regulation?

Answer 19

Allosteric: binding to an enzyme and have the ability to modify it.

Allosteric regulation would mean binding to the site and activating or deactivating the enzyme or increasing or decreasing the enzyme activity.

Question 20

What are the 3 RNA polymerases in eukaryotic transcription?

Answer 20

RNA polymerase 1
RNA polymerase 2
RNA polymerase 3

Question 21

What are the roles of RNA polymerase 1?

Answer 21

Transcription of pre-rRNA (28S, 18S, 5.8S rRNAs)
Ribosome components.
Protein Synthesis.

Question 22

What are the roles of RNA polymerase 2?

Answer 22

Transcription of mRNA - snRNA - siRNA - miRNA.
Encodes proteins.
RNA splicing.
Chromatin-mediated repression.
Translation control.

Question 23

What are the roles of RNA polymerase 3?

Answer 23

Transcription of tRNAs - 5S rRNA - snRNA U6 - 7S RNA - other small stable RNAs.
Protein Synthesis
Ribosome component.
RNA splicing.
Signal recognition particle for insertion of polypeptides into the endoplasmic reticulum.
More unknown functions.

Question 24

Explain the sensitivity of each RNA polymerase to the mushroom toxin α-amnitin.

Answer 24

RNA Pol 1: High sensitivity, very high RNA synthesis.

RNA Pol 2: Lower sensitivity, but still high RNA synthesis.

RNA Pol 3: Lowest sensitivity, low RNA synthesis.

Question 25

Where is situated CTD?

Answer 25

Attached to the C-terminus of the largest subunit of RNA Polymerase 2.

Question 26

What does CTD stand for?

Answer 26

carboxyl terminal domain.

Question 27

What are the common features of the 3 eukaryotic RNA polymerases?

Answer 27

They all exist in multimeric complexes.
They all have a beta’ and a beta subunits, α subunits, ω subunit.
They can show significant similarity to the bacterial subunits.
Most are essential.

Question 28

What are 3D structure differences and similarities between prokaryotic and eukaryotic RNA polymerases

Answer 28

Differences:
Eukaryotic RNA pol 2 has more auxiliary subunits around the large ones.

Similarities
2 large subunits surrounded by auxiliary subunits.
Clamp domain
CTD

Question 29

Why is CTD important?

Answer 29

CTD is essential for survival because of its series of repeats.

Question 30

What’s the role of the Clamp Domain?

Answer 30

Opens to allow the polymerase to interact with DNA.
Once transcription starts, it closes on the DNA, keeping it in place, and that’s how we can transcribe for a long time.

Question 31

When does CTD get phosphorylated?

Answer 31

CTD gets phosphorylated during the initiation stage of transcription.

Question 32

Why is the phosphorylation of CTD interesting?

Answer 32

Because phosphorylated CTD is associated with active transcription. We are able to spot the regions on the chromosomes that are being translated based on where we can find phosphorylated CTD.

Question 33

What are polytene chromosomes and why are we interested in them?

Answer 33

Polytene chromosomes are larger chromosomes. They also have DNA strands lined side by side. They puff during transcription. Since they are bigger, they are visible under a microscope and we can do more and better research with polytene chromosomes.