Lecture 12: Eukaryotic transcription [G] Flashcards
Tuesday 22nd October
What are the differences between bacterial transcription and eukaryotic transcription?
- 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.
Why does mRNA undergo 5’ capping?
To protect from nuclease digestion, and to aid in mRNA export.
Which 3 RNA polymerases are involved in eukaryotic transcription?
RNA polymerase I
RNA polymerase II
RNA polymerase III
Which RNA polymerase is the most important to consider?
RNA polymerase II
What is splicing?
Where non coding introns are removed and the exons are fused together to make one continuous gene
Is it true that the DNA in eukaryotes is tightly packed into chromatin and requires unwinding for transcription?
Yes
What is 3’ polyadenylation?
Where a chain of adenine nucleotides are added to the 3’ end of a newly synthesised mRNA molecule to increase stability
What are the eukaryotic initiation requirements?
- 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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In eukaryotes, do general transcription factors (GTFs) replace the bacterial sigma factor?
Yes
What do sigma factors do?
They identify promoters and attract polymerase.
What does GTF stand for?
General transcription factor
What are most of our promoters called?
TATA box promoters
What sequence do TATA box promoters have?
A TATA box sequence
What is the TATA box sequence?
TATAAAA
Where does the TATA box promoter lie?
Upstream of an initiator element, usually between -30 and -100 from the transcriptional start.
What are located a few kilobases away from the TATA box promoter?
Enhancers,
Are there a such thing as TATA-less promoters?
Yes, and they have a DPE
What other sequences influence the rate of trasncription?
a CAAT box (GGNCAATCT) or a GC (GGGCGG) box positioned between -40 and -150
Explain the superficial similarities between bacterial and eukaryotic replication
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.
Evolutionary links
- 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
How do we unfold higher order structures to allow access of the polymerase to the DNA?
- 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.
What does the TFIID do when it sees a TATA box?
- 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
Initiation
- 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.
What dos TFIIH do?
- 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.
Is it true that the DNA has to be bent in order to get the enhancer close to the promoter?
Yes
Describe enhancers
Binds to mediator proteins, which then interact with the basal transcription complex. DNA looping brings enhancers in proximity to the promoter.
What do silencers do?
- Silencers act similarly to enhancers but recruit repressor proteins, which suppresses transcription.
- Silencers also deacetyle histones, promoting chromatin condensation, and rendering the DNA inacessible.
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)?
Yes
Where are immunoglobins expressed?
Immunoglobulins are only expressed in B cells because the Ig enhancer functions only in B cells.
burkitt’s lymphoma (read more)
- 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.
Initiation to Elongation
- 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.
Is it true that phosphates can alter the function of a protein?
Yes
Elongation
- 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.
Why will eating a death cap mushroom cause death?
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.
What is 5’ capping?
Where the 5’ end is capped by a nucleotide triphosphate through an unusual linkage. The process is accompanied by methylation.
What are the benefits of 5’ capping?
Protects RNA from degradation and aids in ribosome binding.
Describe termination in eukaryotic transcription
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
Describe polyadenylation
- 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.
Describe the chromatin modification of Acetylation
- Adds acetyl groups to histone tails.
- Loosens chromatin, facilitating transcription.
Describe the chromatin modification of Deacetylation
- Removes acetyl groups, condensing chromatin and silencing genes.
Describe the chromatin modification of Methylation
- Adds methyl groups to DNA or histones.
- Often (but not always) represses transcription.
Why is Polyadenylation Important?
- 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.
