Lecture 12: Eukaryotic transcription

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 polymerae 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

What is 3’ polyadenylation?

Answer 6

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

Question 7

What are the eukaryotic initiation requirements?

Answer 7

• 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 8

What do sigma factors do?

Answer 8

They identify promoters and attract polymerase.

Question 9

What does GTF stand for?

Answer 9

General transcription factor

Question 10

What are most of our promoters called?

Answer 10

TATA box promoters

Question 11

What sequence do TATA box promoters have?

Answer 11

A TATA box sequence

Question 12

What is the TATA box sequence?

Answer 12

TATAAAA

Question 13

Where does the TATA box promoter lie?

Answer 13

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

Question 14

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

Answer 14

Enhancers,

Question 15

Are there a such thing as TATA-less promoters?

Answer 15

Yes, and they have a DPE

Question 16

What other sequences influence the rate of trasncription?

Answer 16

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

Question 17

EXplain the superficial similarities between bacterial and eukaryotic replication

Answer 17

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.

Answer 18

• 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 19

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

Answer 19

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 20

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

Answer 20

• It stops and binds to the TATA box. This initiates transcription. When the TFIID binds, it bends the DNA and widens the groove

Question 21

Initiation

Answer 21

• 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.

• 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 22

What dos TFIIH do?

Answer 22

It acts as both a helicase (unwinds DNA) and kinase (phosphorylates RNA polymerase II).

Question 23

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

Answer 23

Yes

Question 24

Describe enhancers

Answer 24

Binds to mediator proteins, which then interact with the basal transcription complex. The DNA becomes looped and transcription is boosted.

Question 25

What do silencers do?

Answer 25

Silencers act similarly but recruit repressors, reducing gene expression.

Question 26

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 26

Yes

Question 27

Why are immunoglobins expressed?

Answer 27

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

Question 28

burkitt’s lymphoma (read more)

Answer 28

If MYC is over or under-expressed, results in cell proliferation and genome instability.

Question 29

Initiation to Elongation

Answer 29

• 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

Question 30

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

Answer 30

Yes

Question 31

Elongation

Answer 31

• Phosphorylation and change in function of the C terminal domain
• Dissociation of TFIIB, E and H from the basal transcription apparatus.
• This leaves TFIIF associated with the polymerase.
• TFIIF converts the polymerase into a highly processive machine, that moves along the DNA, and copies RNA.
• The promoter and TFIID/A complex are freed from the promoter,

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Question 32

Why will eating a death cap mushroom cause death?

Answer 32

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 33

What is 5’ capping?

Answer 33

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

Question 34

Describe termination in eukaryotic transcription

Answer 34

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