Lecture 12: Eukaryotic transcription [G]

Question 1

What are the differences between bacterial transcription and eukaryotic transcription?

Answer 1

• In bacteria, there is no nucleus separating the DNA from the cytoplasm, so transcription and translation occur simultaneously. Whereas eukaryotes have a nucleus, so transcription occurs in the nucleus and translation occurs in the cytoplasm.
• In bacteria, mRNA is used directly without modification. However, eukaryotic mRNA receives extensive processing, such as: 5’capping, splicing, and polyadenylation.
• Bacteria use a single RNA polymerase to transcribe genes. Whilst eukaryotes use 3 different types of polymerases.
• In eukaryotes, the DNA is tightly wrapped around nucleosomes and must be unwound before transcription can occur. This isn’t the case for prokaryotes.
• The promoters in bacteria are less complex, whilst the promoters in eukaryotes are more complex, involving transcription factors and regulatory elements like enhancers.

Question 2

Why does mRNA undergo 5’ capping?

Answer 2

To protect from nuclease digestion, and to aid in mRNA export.

Question 3

Which 3 RNA polymerases are involved in eukaryotic transcription?

Answer 3

RNA polymerase I

RNA polymerase II

RNA polymerase III

Question 4

Which RNA polymerase is the most important to consider?

Answer 4

RNA polymerase II

Question 5

What is splicing?

Answer 5

Where non coding introns are removed and the exons are fused together to make one continuous gene

Question 6

Is it true that the DNA in eukaryotes is tightly packed into chromatin and requires unwinding for transcription?

Answer 6

Yes

Question 7

What is 3’ polyadenylation?

Answer 7

Where a chain of adenine nucleotides are added to the 3’ end of a newly synthesised mRNA molecule to increase stability

Question 8

What are the eukaryotic initiation requirements?

Answer 8

• Eukaryotic transcription has a highly packed substrate, containing structures that need to be unwound prior to transcription.
• Whilst bacterial RNA polymerase only requires a sigma factor, eukaryotic RNA polymerases require multiple additional genes called the general transcription factors.

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Question 9

In eukaryotes, do general transcription factors (GTFs) replace the bacterial sigma factor?

Answer 9

Yes

Question 10

What do sigma factors do?

Answer 10

They identify promoters and attract polymerase.

Question 11

What does GTF stand for?

Answer 11

General transcription factor

Question 12

What are most of our promoters called?

Answer 12

TATA box promoters

Question 13

What sequence do TATA box promoters have?

Answer 13

A TATA box sequence

Question 14

What is the TATA box sequence?

Answer 14

TATAAAA

Question 15

Where does the TATA box promoter lie?

Answer 15

Upstream of an initiator element, usually between -30 and -100 from the transcriptional start.

Question 16

What are located a few kilobases away from the TATA box promoter?

Answer 16

Enhancers,

Question 17

Are there a such thing as TATA-less promoters?

Answer 17

Yes, and they have a DPE

Question 18

What other sequences influence the rate of trasncription?

Answer 18

a CAAT box (GGNCAATCT) or a GC (GGGCGG) box positioned between -40 and -150

Question 19

Explain the superficial similarities between bacterial and eukaryotic replication

Answer 19

① the spacing is different, and the CAAT box (or GC box) of eukaryotic promoters can be on EITHER strand.

② the -10 and -35 sequences interact with a part of the holoenzyme (σ), whereas the TATA, CAAT and GC motifs are recognised by other proteins, and not RNA Pol II.

Question 20

Evolutionary links

Answer 20

• Both archaea and eukaryotes have internal membranes
• We both have TATA box promoters (suggests that our nuclei is derived from archaeas)
• Our ribosomes are more similar to archaeal ribosomes than to bacterial ribosomes
• Our cytosol is also largely derived from archaea
• Our mircohondria comes from free living bacteria
• tRNAs are transcribed as individual genes in both archaea and eukaryotes
• Whilst archaea have only one polymerase, it looks a lot like eukaryotic polymerase, and not bacterial

Question 21

How do we unfold higher order structures to allow access of the polymerase to the DNA?

Answer 21

• The DNA is wrapped around nucleosomes, each with 8 subunits. The nucleosomes have long tails that stick out from them.
• The acetylation of histone tails promotes a loose chromatin structure that permits transcription.

Question 22

What does the TFIID do when it sees a TATA box?

Answer 22

• It contains the TATA-binding protein (TBP), so it stops and binds to the TATA box. This initiates transcription. When the TFIID binds, it bends the DNA and widens the groove

Question 23

Initiation

Answer 23

• The TATA box binding protein from TFIID binds to the TATA box promoter.

-TFIIA then binds to one part of the TATA box binding protein, , stabilizing the TBP-DNA interaction.

• This then recruits TFIIB. This binds to both sides of the TATA box
• TFIIB then recruits TFIIF, RNA polymerase, TFIIE, and TFIIH.
• Once this whole complex is formed, it will stay locked on the TATA box promoter. This complex is known as the basal transcription apparatus.
• Additional elements, such as enhancers, will then be required to get the complex moving.

Question 24

What dos TFIIH do?

Answer 24

• It acts as both a helicase (unwinds DNA) and kinase (phosphorylates RNA polymerase II).
• Helicase activity: Unwinds the DNA at the transcription start site to create the transcription bubble.
• Kinase activity: Phosphorylates the C-terminal domain (CTD) of RNA Polymerase II, transitioning it from initiation to elongation.

Question 25

Is it true that the DNA has to be bent in order to get the enhancer close to the promoter?

Answer 25

Yes

Question 26

Describe enhancers

Answer 26

Binds to mediator proteins, which then interact with the basal transcription complex. DNA looping brings enhancers in proximity to the promoter.

Question 27

What do silencers do?

Answer 27

- Silencers act similarly to enhancers but recruit repressor proteins, which suppresses transcription. - Silencers also deacetyle histones, promoting chromatin condensation, and rendering the DNA inacessible.

Question 28

is is true that the presence of enhancers allows for cell-specific control of gene expression, stopping, for example, liver cells making crystallin (a component of the eye lens)?

Answer 28

Yes

Question 29

Where are immunoglobins expressed?

Answer 29

Immunoglobulins are only expressed in B cells because the Ig enhancer functions only in B cells.

Question 30

burkitt's lymphoma (read more)

Answer 30

- Caused by chromosomal translocation of the MYC oncogene near the immunoglobulin heavy chain promoter. - Results in uncontrolled MYC expression, promoting rapid cell division and genomic instability.

Question 31

Initiation to Elongation

Answer 31

- TFIIH is both a helix that opens the DNA double helix, and also a kinase that phosphorylates the C'- terminal domain of the RNA Pol II L’ subunit. - This phosphorylation is what marks the transition from initiation to elongation - The transcription bubble forms, and RNA synthesis begins.

Question 32

Is it true that phosphates can alter the function of a protein?

Answer 32

Yes

Question 33

Elongation

Answer 33

- Phosphorylation of the CTD initiates elongation by altering the function of RNA Polymerase II. - TFIIB, TFIIE, and TFIIH dissociate from the transcription complex. - TFIIF remains associated with the transcription complex, stabilizing RNA Polymerase II and ensuring processivity. - RNA Polymerase II moves along the DNA, unwinding the template and synthesizing RNA. - The TFIID/TFIIA complex may remain at the promoter to facilitate re-initiation or dissociate as needed.

Question 34

Why will eating a death cap mushroom cause death?

Answer 34

Because the death cap contains α-amanitin, which is a potent elongation inhibitor of RNA polymerase II. So transcription can't occur in organs such as the liver and the kidneys.

Question 35

What is 5' capping?

Answer 35

Where the 5’ end is capped by a nucleotide triphosphate through an unusual linkage. The process is accompanied by methylation.

Question 36

What are the benefits of 5' capping?

Answer 36

Protects RNA from degradation and aids in ribosome binding.

Question 37

Describe termination in eukaryotic transcription

Answer 37

① The rate of transcription of RNA Pol II slows down in the 3’ UTR (3’ untranslated region) ② The RNA is cleaved, and a poly(A) tail is added. ③ The RNA fragment in contact with polymerase is degraded, Pol II changes conformation and disengages from the DNA

Question 38

Describe polyadenylation

Answer 38

- As RNA Polymerase II transcribes the gene, it encounters a conserved sequence in the pre-mRNA near the 3′ end. This sequence is usually AAUAAA or a variant, followed by a GU-rich region downstream. - The transcription machinery slows down near the polyadenylation signal. Specialized proteins, called cleavage factors, bind to the signal and cut the RNA just downstream of it. - An enzyme called poly(A) polymerase (PAP) adds a tail of ~50–250 adenine nucleotides to the cleaved 3′ end. - Poly(A)-binding proteins bind to the poly(A) tail, stabilize the tail and regulate RNA degradation. - During translation, the poly(A) tail interacts with the 5′ cap to form a circularized mRNA structure, enhancing the efficiency of ribosome recruitment.

Question 39

Describe the chromatin modification of Acetylation

Answer 39

- Adds acetyl groups to histone tails. - Loosens chromatin, facilitating transcription.

Question 40

Describe the chromatin modification of Deacetylation

Answer 40

- Removes acetyl groups, condensing chromatin and silencing genes.

Question 41

Describe the chromatin modification of Methylation

Answer 41

- Adds methyl groups to DNA or histones. - Often (but not always) represses transcription.

Question 42

Why is Polyadenylation Important?

Answer 42

- Increases Stability: The poly(A) tail prevents the mRNA from being degraded too quickly by cellular enzymes. - Regulates Translation: Length of the tail can affect how actively the mRNA is translated into protein. - Enables Nuclear Export: Properly polyadenylated mRNAs are recognized for export from the nucleus to the cytoplasm.