7) Eukaryotic Transcription

Question 1

What are the 7 important facts about eukaryotic transcription? (Sentences only)

Answer 1

1) Separation of transcription and translation
2) More than one RNA polymerase
3) Transcription factors
4) Extra proteins associate with holoenzyme
5) The Mediator complex
6) Transcript processing
7) Histones regulate access to promoters

Question 2

1) How is eukaryotic transcription and translation separated?

Answer 2

SPATIAL separation= Does not happen in prokaryotes as they do not have ORGANELLES

Eukaryotic:
Nucleus= When transcription happens first but can then go onto using different organelles

DNA is replicated in eukaryotic nucleus then transcribed and mRNA is exported from the nucleus into the cytoplasm

Question 3

2) What are the different RNA polymerases involved in eukaryotic transcription? (Names ONLY)

Answer 3

Prokaryotes: ONLY have 1 RNA polymerase

Eukaryotes: More than one RNA polymerase but all have the same structure but have different proteins and roles

RNA polymerase I
RNA polymerase II
RNA polymerase III

Question 4

2) For each RNA polymerase in eukaryotic transcription, where is it found? What does it produce? What is its a-amanitin sensitivity?

Answer 4

RNA polymerase I:
Where: Nucleolus
Synthesises: rRNA
A-amanitin sensitivity: None

RNA polymerase II:
Where: Nucleoplasm
Synthesises: hnRNA (protein coding genes)
A-amanitin sensitivity: High

RNA polymerase III:
Where: Nucleoplasm
Synthesises: tRNA, other small nuclear RNAs
A-amanintin sensitivity: Medium

Question 5

2) What is alpha aminitin and how can it be used to distinguish between the 3 polymerases?

Answer 5

Alpha aminitin: Fungal toxin
Distinguish: Each has different sensitivities to it

For Pol I: Activity is unchanged when toxin is added as it is not sensitive to it at all

For Pol II: Activity complete decreases as sensitivity is high

For Pol III: Activity is somewhere between as is medium sensitivity

Question 6

3) What are transcription factors?

Answer 6

Transcription of protein coding genes requires transcription factors

Euk: Positive and negative regulatory proteins
-Bind to the promoter of protein coding genes
EUKARYOTES: USE POSITIVE FACTORS (activators) MORe than repressors + transcription cannot happen without them

Transcription factors: Regulatory factors whether positive or negative

Many different ones, and there is one for each environment or developmental condition which organism needs to respond by switching on gene expression

Question 7

3) What do transcription factors bind to?

Answer 7

Bind to recognition sequences in the promoter= Promoter motifs or cis-acting elements

Unlike in prokaryotes, motifs can be far away form the gene yet influence its expression

Transcription factor + Cis-element= RNA Pol II can work

Question 8

4)What are the extra proteins associated with holoenzyme?

GTFs

Answer 8

GTFs= General Transcription Factors
Needed for every gene
Function: Position Pol II in right place and help separate strands so the template can get into the active site of the enzyme

Pol II + GTFs= Preintiation complex

Similar function to sigma factors in prokaryotes

However: Just these two alone do not start transcription, still needs something to activate the activator (Mediator transcriptional co-activator complex)

Question 9

5) What is the mediator transcriptional co-activator?

Answer 9

Mediator transcriptional co-activator:
Co-activator: Needed along with activators (transcription factors) to switch on transcription

Mediates: Links transcription factors to GTFs and RNA Pol II

MOLECULAR BRIDGE

Question 10

5) What is the structure like for the mediator and how does it bind in yeast?

Answer 10

Head
Kinase on top of Middle part
Tail

In yeast: Tail binds to Transcription Factors
Head: Binds Pol II
25-35 proteins, all act together as a complex

Kinase: 4 extra proteins that regulates the activity as whole

Question 11

5) How does the mediator complex bring Pol II and Tfs close together?

Answer 11

Causes DNA to loop
Allows: Transcription factors and their cis elements (promoter motifs) into close proximity with Pol II
Result: Pol II can transcribe the gene

Question 12

6) Why does the transcript processed in eukaryotic transcription?

Answer 12

Euk: Hetronuclear unprocessed RNA= Needs to be processed in order for it to be transcripts that can be translated

INTRONSSSSSSSSSS= Prokaryotes do not have these as each coding sequence within the mrNA is translated quite separately (polycistronic mRNA)

Question 13

6) What is the difference between mRNA within prokaryotes and eukaryotes?

Answer 13

mRNA= Only contains one coding sequence, often interrupted by introns

Introns are removed by splicing
After transcription has finished (post-transcriptional), further modifications to mRNA
5’-CAP (Modified quanicine residue) is added and polyA-tail (3’ end)

Polymerase II synthesises RNA whilst co-ordinating processing at the same time so it needs to have all these functions

Question 14

6) What are the 3 modifications needed to be made to mRNA before it can be translated?

Answer 14

1) Introns should be spliced= Only left with coding regions
Occurs in the nucleus with aid of many snRNA and their associated proteins, which can recognise exactly where the exon-intron boundaries are
HOW: Specific adenine attacks the boundary on the 5’ side of the intron and causes cleavage= Free intron end to which the adenine can become covalently bound + Further snRNA initiates the formation of the lariat and promotes the intro excision

2) 5’-CAP added by proteins that interact with CTD (C-terminal domain of RNA polymerase)= Addition of GTP molecule to the 5’ end of the mRNA. GTP is further modified by addition of a methyl group, CAP can only be added to diphosphate or a triphosphate group
IF mRNA was broken in middle, only a monophosphate would be present at the 5’ end= No Cap= RNA molecule wouldn’t be recognised as a transcript

Function of CAP= Prevents RNA degradation + Identifies that shows this molecule is mRNA

3) Addition of a poly A tail
Addition of “AAAAAAAA” to 3’ end
Function: Helps to stabilise the mRNA, signals the point where the mRNA must be cut off

Functions are coordinated by Pol II’s essential C-terminal domain repeat

Question 15

6) How is hnRNA is processed speedily and efficiently?

Answer 15

Eukaryotic RNA polymerase has coordinating functions

Functions are coordinated by Pol II’s essential C-terminal domain repeat which is responsible for regulating transcription but also recruits factors that aid in 5’ CAP addition, splicing and polyA tail

Basically: Pol II can do everything.

Question 16

7) How can histones regulate access to promoters?

Answer 16

Nucleosomes= 4 types of histones

Chemical modifications of histones= Cell modulates nucleosome mobility and turnover

Histone modifications= Control whether DNA can be transcribed

Number of enzymes that add or remove chemical groups onto or from the N terminal protein tails of histones= Regulate the histone’s ability to bind to the DNA tightly or not

Resulting histone tails= Way of regulating access to the DNA by Pol II

Small chemical modifications which tells whether to not the nucleosomes will be tightly wound= Influences the movement of the tail and winding and unwinding

Question 17

7) How does histone acetylation regulate transcription?

Answer 17

Acetylation: Adding acetyl groups to histone tails

Targets Lys residues in amino-terminal tails of core histone proteins

Acetylation= Inhibits the folding of nucleosome arrays into secondary and tertiary chromatin structures

Histone tail acetylation= Chromatin decondensation= Allowing access to transcription factors

Acetylated= Loose winding