Transcription in Eukaryotic - Week 4

Question 1

how is transcription different in eukaryotes and bacteria

Answer 1

The transcription in eukaryotes is more complex than in bacteria, some of the key main differences are:
- eukaryote cells contain 3 different RNA polymerases, (the 3 different RNA polymerases are able to transcribe distinct categories of the genes), whereas bacterial cells only contain 1 RNA polymerase
- eukaryotic polymerases need to interact with additional proteins to initiate transcription rather than binding directly to the promoter sequence as in bacteria.

Question 2

what are the additional proteins and regulatory elements does eukaryotic RNA polymerase need to integrate with to initiate transcription

Answer 2

These additional proteins are called general transcriptional factors and there are 5 different types.
RNA polymerases also interact with additional regulatory elements which are called enhancers, silencers, chromatin modifiers and etc.

Question 3

what are the 3 different types of eukaryotic RNA polymerase and what are they responsible for

Answer 3

The 3 different RNA polymerases are:
- RNA polymerase I
Which is responsible for the synthesis of all ribosomal RNAs, except for the 5S ribosomal RNA.
- RNA polymerase II
Which is responsible for the synthesis of all mRNA (messenger RNA).
- RNA polymerase III
Which is responsible for the synthesis of all tRNA (transfer RNA) and 5S ribosomal RNA.

RNA polymerase II and III are also involved in the synthesis of ssRNA (small nuclear RNA) which is important for the regulation of gene expression.

Question 4

what is the function of mitochondrial RNA polymerase

Answer 4

RNA polymerases in the mitochondria are responsible for the transcription of mitochondrial genes.

Question 5

what is the function of RNA polymerase in the chloroplast

Answer 5

RNA polymerases in the chloroplast are responsible for the transcription of chloroplast genes.

Question 6

what is the similarity between prokaryotic/bacteria polymerase and eukaryotic polymerase

Answer 6

Both eukaryotic and bacterial RNA polymerases have core catalytic subunits that are responsible for synthesizing RNA from a DNA template. In bacteria, the core enzyme consists of five subunits: two α subunits, one β subunit, one β’ subunit, and one ω subunit. In eukaryotes, the core enzyme consists of 12 subunits, including homologs of the bacterial α, β, and β’ subunits.

Question 7

what is the difference between prokaryotic/bacteria polymerase and eukaryotic polymerase

Answer 7

Eukaryotic RNA polymerases recognize complex promoter sequences, often with the help of additional transcription factors, while bacterial RNA polymerases recognize relatively simple promoter sequences called “sigma factors.” This difference in promoter recognition requires eukaryotic RNA polymerases to have additional subunits and mechanisms for promoter recognition, which are not present in bacterial RNA polymerases.

Question 8

which subunit is found in all 3 RNA polymerases

Answer 8

the subunit that is found in RNA polymerase I, II and III is RPB6

Question 9

how many subunits does RNA polymerase II consist of and which is the largest

Answer 9

Polermase II consists of 12 subunits and cannot initiate transcription by itself as it needs transcription factors to initiate transcription.

The largest subunit of Pol II has a carboxy-terminal domain (CTD), which consists of multiple repeats of 7 amino acids. CTD is subjected to phosphorylation and is important for initiation, elongation and all aspects of mRNA processing.

Question 10

what are the transcription factors polymerase ll needs and why are they important

Answer 10

Pol II needs a set of transcription factors which are:

• General transcription factors (GTF) – important for initiation by Pol II. They are identified as TFNX.
• Regulatory transcription factors – important for regulating gene expression.

Question 11

what is a promoter

Answer 11

The promoter is a short DNA sequence that is required for transcription to take place.

Question 12

what 2 sequences are found in the core promoter

Answer 12

In eukaryotes, the core promoter contains 2 important sequences, which are:
- TATA box
- initiator box

Question 13

where is the TATA box located and why is it important

Answer 13

The TATA box is a sequence that is located about -25 base pairs upstream from the transcription start site.
This TATA box is important for the binding of RNA polymerases.

Question 14

what is the initiation site and why is it important

Answer 14

The initiation site is where the transcription starts. If the initiation site is missing from the core promotor, the transcription can start at the wrong location. Therefore it is an important site for the initiation of the transcription at the right place.

Question 15

where are regulatory elements found and what is their function

Answer 15

Regulatory elements are found several thousand bp upstream of the start site. They affect the ability of RNA polymerase to recognize the core promoter and begin transcription.

Question 16

what are the 2 types of regulatory elements and what is their function

Answer 16

There are two types of regulatory elements:
- activating sequences which are called Enhancers
- inhibiting sequences which are called Sliencer elements
These 2 distinct elements have different functions
The enhancer elements promote transcription whereas the silencer elements inhibit the transcription, thus both these elements affect the rate of transcription.

Question 17

why are trans-acting factors called trans-acting factors

Answer 17

the regulatory transcription factor is also called the trans-acting factor because while the enhancer elements and the silencer elements are located within the chromosome of the genes that they regulate, the genes that transcribe the trans-acting factors are located on different chromosomes.

Question 18

what are the 6 general transcription that RNA polymerase II requires for the initiation of transcription

Answer 18

Six general transcription factors are required for the initiation of transcription by RNA Pol II.
RNA polymerase II requires 6 general transcription factors, which are:
- TFIID
- TFIIA
- TFIIB
- TFIIF
- TFIIE
- TFIIH

( TF stand for transcription factor, II stands for RNA polymerase II)

Question 19

what are the different general transcription factors and how many subunits does each general transcription factor have

Answer 19

GTFs Number of subunits
TBP 1
TFIIA 2
TFIIB 1
TFIIE 2
TFIIF 3
TFIIH 10
TAFs 11

Question 20

why are the 6 transcription factors required

Answer 20

The 6 general transcription factor is required for the initiation of transcription by RNA polymerase II, there are a series of interactions between the 6 general transcription factors and RNA polymerase II which results in the formation of the open complex or in the result in the formation of a pre-initiation complex.

Question 21

which transcription factor bind to the TATA box and in which order

Answer 21

The first transcription factor that binds to the TATA box is TFIID (TFIID is composed of several subunits which also include the TATA-binding protein which is TBP).
After TFlID binds to the TATA box, TFIIB joins the complex, (the function of TFIIB is to promote the binding of the RNA polymerase II to the promoter).
RNA polymerase II is already associated with TFIIF, so together they join to make the complex.
The last 2 general transcription factors that join the complex are TFIIE and TFIIH.

When all the general transcription factor is associated with the RNA polymerase II and they assemble at the promoter, they form the closed complex.

Question 22

what happens after the general transcription factor binds to RNA polymerase II

Answer 22

When all the general transcription factor is associated with the RNA polymerase II and they assemble at the promoter, they form the closed complex.
Then the TFIIH (which is a multisubunit factor) unwinds the DNA locally to form the transcription bubble. Essentially TFIIH acts as a helicase that unwinds the DNA at the initiation site.

Question 23

Why is TFIIH important other than the fact it’s able to unwind DNA

Answer 23

TFIIH is also able to phosphorylate the RNA polymerase II into the carboxyl-terminal domain (CTD), this phosphorylation event is important because it marks the transition from the initiation step to the elongation step of the transcription.

Question 24

why is the phosphorylation of RNA polymerase II by TFIIH important

Answer 24

TFIIH is a multi-subunit protein complex that plays a crucial role in transcription initiation by serving as a DNA helicase and a kinase.
phosphorylation of RNA polymerase II by TFIIH is a critical step in the regulation of transcription initiation in eukaryotic cells, facilitating the recruitment of additional factors and influencing the transition from initiation to elongation during transcription.

Question 25

what is promoter clearance and how does the elongation step start

Answer 25

After the phosphorylation of RNA polymerase II by TFIIH, RNA polymerase II undergoes conformational changes and escapes from the promoter region, resulting in promoter clearance. Once RNAPII has cleared the promoter, it enters the transcription elongation phase.
So after RNA polymerase II clears the promoter, the elongation step starts.

Question 26

what happens in the elongation step

Answer 26

During transcription elongation, RNA polymerase II moves along the DNA template strand and synthesizes an RNA transcript that is complementary to the DNA template. The elongation step of transcription involves the continuous addition of nucleotides to the growing RNA chain, while RNAPII moves along the DNA template strand. Essentially, it starts to add nucleotides to the growing RNA chain, it starts to add one nucleotide at a time to the growing RNA chain.

Question 27

what is the melted region and transcription bubble in transcription

Answer 27

During transcription, RNA polymerase II unwinds the DNA double helix at the transcription start site to form a transcription bubble. This allows the RNA polymerase to access the template DNA strand and synthesize an RNA molecule using complementary base pairing. The region of unwound DNA ahead of the transcription bubble is called the “melted region” or “open complex,” and it provides the template for RNA synthesis.

Question 28

when does the transcription bubble close and why is it important

Answer 28

Once the transcription is initiated, RNA polymerase II moves along the template DNA strand, synthesizing an RNA molecule in the 5’ to 3’ direction. As the RNA polymerase moves forward, it continues to unwind the DNA ahead of the transcription bubble, creating a stable transcription elongation complex. After the RNA polymerase has passed through a particular region of DNA, the transcription bubble closes behind it, allowing the DNA double helix to re-form. This process helps to maintain the integrity of the DNA double helix and ensures that the transcription process can proceed efficiently.

Question 29

what is the final stage of transcription and when does it happen

Answer 29

The final stage of transcription is termination.
Termination occurs when a transcribing RNA polymerase releases the DNA template and the nascent RNA. RNA polymerase releases the DNA template and RNA when the RNA polymerase reaches the transcription terminator sites, which are specific sequences on the DNA template.

However very little is known about transcription termination in eukaryotic systems.

Question 30

what termination mechanisms do the 3 different polymerases use

Answer 30

Three Eukaryotic RNA Polymerases employ different termination mechanisms:
- Pol I uses a combination of DNA sequences and DNA-binding proteins
- Pol II couples transcription termination to cleavage and poly-adenylation of the nascent transcript’ 3” end
- Pol III uses RNA sequences

Question 31

what are the 2 models of termination that have been proposed in eukaryotes

Answer 31

There are 2 models of termination that have been proposed in eukaryotes, which are:
- Anti-terminator model (aka allosteric model)
- Torpedo model

Question 32

when does RNA transcript have modifications

Answer 32

In eukaryotes, transcription initially produces a longer RNA, which is called primary RNA transcript (aka immature RNA transcript).
This primary RNA transcript undergoes modifications in the nucleus before it exits the nucleus.
The final product is called mature messenger RNA (mRNA).

Question 33

what are the 3 modifications the primary RNA go through

Answer 33

This primary RNA transcript undergoes 3 modifications in the nucleus. Which are:
1. Modification at the 5’ end, which is called 5’ capping
2. Modification at the 3’ end which is the poly(A) tail.
3. The final modification is splicing.

Question 34

what happens when the modifications are complete

Answer 34

After all the RNA modifications are completed, the mRNA leaves the nucleus and enters the cytosol, where it is ready for translation.

Question 35

what is 5’ end modification

Answer 35

The first modification in the immature mRNA is at the 5’ end, called the 5’ end-capping.
After the RNA polymerase II synthesises the transcript so when the transcript is about 20 nucleotides in length, a modified form of guanine is covalently attached at the 5’ end. This event is called 5’ capping. The capping is an enzymatic reaction catalysed by a specific nuclear enzyme called guanylyl-transferase (GT).

Question 36

what is the function of the 5’ cap

Answer 36

the 5’ cap has 3 important functions, which are:
- it prevents the degradation of the mRNA
- it is required for the transport of mRNA out of the nucleus
- it is required for the initiation of translation

Question 37

what is splicing and what are exons

Answer 37

Another modification of the immature RNA (primary RNA) is splicing
Splicing involves the removal of segments called introns and adjacent exons are then covalently joined. The segments that are retained in mature RNA are called exons.
Therefore exons are protein-coding sequences as introns are deleted during splicing.

Question 38

what chemically happens at the capping event of the 5’ end

Answer 38

The capping event is an enzymatic reaction. The capping event occurs while the primary mRNA is being made by RNA polymerase II.
what happens at the 5’ capping event is the 7-methyl-guanosine is attached to the 5’ end of the newly transcribed mRNA and the linkage between the cap and mRNA is a 5’ end to 5’ end linkage.

Question 39

what is the poly-A tail and how is it added

Answer 39

Mature eukaryotic mRNAs have a tail, which is a string of adenine nucleotides. Usually, the string of nucleotides is 200 adenine in length. This string of adenine nucleotides is known as the poly(A) tail. Most eukaryotic mRNAs have a poly(A) tail at the 3’ end.
This poly(A) tail is not encoded in the gene sequence. This tail is added by an enzyme and is catalysed by a specific enzyme that is called Poly(A) polymerase (PAP).

Question 40

how is the modification of the 3’ end of the transcript made

Answer 40

The modification at the 3’ end of the transcript is made by cleavage and the addition of usually 200 nucleotides (adenine nucleotides) by an enzyme called PAP.

The polyadenylation event occurs in 2 steps:
1- first, when the RNA polymerase II transcribes a consensus sequence, there is an endonuclease that cuts the RNA around 20 nucleotides downstream from the consensus sequence and thus makes the pre-mRNA shorter at the 3’ end.
2- then there is the enzyme PAP that adds adenine

Cleavage of the 3’ end occurs from the transcription complex by a Clipping enzyme

Question 41

what is the function of the poly-A tail

Answer 41

The function of the poly A tail is to protect the 3-end from nuclease degradation and to enhance translatability.

Question 42

what does the torpedo model suggest

Answer 42

This model suggests that RNA polymerase II is physically removed from the DNA template. What happens is
- RNA polymerase transcribes the Poly-A signal, and the transcript is released. But the RNA polymerase II is still associated with the template and still transcribes new transcript but this transcript does not contain the 5’ end cap and is recognised by an exonuclease that is called Rat1.
-This exonuclease degrades the newly transcribed RNA without the 5’ end cap faster than the transcript is synthesised by RNA polymerase.
- The exonuclease at some point catches up to RNA polymerase and this causes RNA polymerase to dissociate from the DNA template.

side note - This model is similar to how bacterial transcription termination factor Rho functions