Regulation of gene expression in eukaryotes

Question 1

What was the original view for what happens to genes when a cell differentiates and what is the contemporary view?

Answer 1

Original view - genes selectively lost when a cell differentiates
Contemporary view - differentiation depends on changes in gene expression (evidence from Dolly the sheep -

Question 2

What are the main ways in which gene expression can be regulated in eukaryotes?

Answer 2

Transcriptional control (most important)
RNA processing control
RNA transport control
mRNA degradation control
Translational control

Question 3

What are transcription factors?

Answer 3

Proteins that recognise and bind to specific DNA sequences in regulatory regions of genes to regulate the onset of transcription

Question 4

What do DNA binding proteins do? (DNA binding proteins involved in transcription are transcription factors)

Answer 4

Distort DNA structure by causing bending/looping that make it more accessible to RNA polymerase
Have structural motifs that can ‘read’ sequences

Question 5

What is the simplest DNA binding motif and where can it be found in prokaryotes and eukaryotes?

Answer 5

The helix-turn-helix motif
In prokaryotes - eg. trp repressor
In eukaryotes - eg. homeodomain
(this is two helices held at a fixed angle, the carboxy terminal helix is a recognition helix that fits into a major groove, amino acid side chains also recognise DNA binding sequence)

Question 6

How can DNA/protein interactions be measured?

Answer 6

Using Electrophoretic Mobility Shift Assay (EMSA) - radioactively labelled DNA is exposed to proteins then gel electrophoresis carried out to see speed
Binding of proteins will slow movement and so shift bands, allowing you to see mobility shift

Question 7

What are the two ways in which transcription factors can control transcription?

Answer 7

Positive and negative regulation

Question 8

What is the simplest example of a genetic switch?

Answer 8

The E. coli tryptophan repressor
Tryptophan can be made in cell or taken up from environment so when available in medium, it is not required to be made
When trp is present it enters cell and trp operon is switched off

Question 9

How is the trp operon switched off in the presence of trp?

Answer 9

Tryptophan acts as a corepressor and binds to a trp repressor causing a conformational shape change so that the repressor (a helix-turn-helix protein) can bind to a recognition sequence in the operator and switch the operon off
(in this way the repressor is activated in order to switch the operon off)

Question 10

What is an operator?

Answer 10

A short DNA sequence that proteins controlling transcription can bind to (activators and repressors)

Question 11

Who extensively studied the lac operon in E.coli?

Answer 11

Jacob and Monod

Question 12

How is the lac operon positively regulated?

Answer 12

By the CAP activator (a helix-turn-helix protein) - switches on operon if glucose is absent (as glucose preferred carbon source)
At low glucose levels, cAMP levels are high, cAMP binds to a cAMP receptor protein (also known as CAP - catabolite activator protein)
CAP has conformational shape change so can bind to CRP binding site near promoter region
Assists in binding RNA polymerase due to protein-protein interactions with polymerase so transcription initiated

Question 13

How is the lac operon negatively regulated?

Answer 13

By the lac repressor - switches operon off if lactose not present
If allolactose (a lactose isomer, acts as an inducer) binds to lac repressor which alters the orientation of headpieces so it no longer fits to the operator of DNA and so transcription is no longer inhibited

Question 14

What is an inducer?

Answer 14

A molecule that inactivates a repressor protein via allosteric interactions causing a conformational shape change so reducing affinity for operator meaning more transcription can occur

Question 15

How is gene expression different in eukaryotes to prokaryotes?

Answer 15

General transcription factors are needed to activate RNA polymerase II
Spatial and temporal separation of transcription and translation so more opportunities for control
(these can act even when bound to DNA many Kb’s away from the RNA polymerase binding site)

Question 16

What is an enhancer?

Answer 16

Regulatory DNA sequences that transcription factors bind to, can be in either orientation
DNA loops out so proteins bound can interact with TFs/polymerase

Question 17

What is a promoter?

Answer 17

A DNA sequence which RNA polymerase and general transcription factors can bind to

Question 18

How is the RNA polymerase II complex assembled?

Answer 18

TBP subunit of TFIID binds TATA box (this unwinds DNA so promotes binding of other proteins)
TFIIB enters complex then RNA polymerase II enters complex escorted by TFIIF
TFIIE and TFIIH also assemble into complex
TFIIH phosphorylates C-terminal domain of polymerase with ATP which releases the polymerase so it can initiate transcription

Question 19

What two domains do transcription factors have?

Answer 19

A DNA binding domain (eg. helix-turn-helix)
An activation domain (eg. acidic domains, glutamate rich, proline rich)
(these domains are independent so domain swap experiments are possible)

Question 20

How do activation domains of transcription factors work?

Answer 20

Accelerate assembly of basal machinery (eg. promoting entry of TFIIB into complex or loading TFIID onto DNA)
Enhance stability/activity of basal transcription machinery

Question 21

What are possible theories for how repressor mechanisms work?

Answer 21

Not well understood - could involve competitive DNA binding, masking activation surface of activators or direct interaction with basal machinery

Question 22

What is promoter-proximal pausing of RNA polymerase II?

Answer 22

Where the polymerase is paused after transcribing around 25 nucleotides so that synthesis can occur rapidly once triggered
Eg. heat-shock induces activation of the heat-shock transcription factor which stimulates paused polymerase to continue chain elongation

Question 23

What does RNA processing involve?

Answer 23

Capping, cleavage, polyadenylation, splicing (all happen in nucleus)
Pre-mRNA transcripts are associated with a class of RNA-binding proteins called hnRNPs

Question 24

What is capping of mRNA?

Answer 24

A 7-methylguanosine cap is added to the 5’ end of transcript by a capping enzyme that associates with phosphorylated C-terminal domain of polymerase after transcription initiation
The cap is required for efficient nuclear export and translation

Question 25

What is cleavage/polyadenylation of mRNA?

Answer 25

There is a conserved polyadenylation signal 10-30 nucleotides upstream from poly(A) site where cleavage and polyadenylation occur
A multiprotein complex including poly(A) polymerase (PAP) carries out cleavage and polyadenylation
PABII (a polyA binding protein) stimulates addition of A residues by PAP then stops addition once polyA tail is 200-250 residues long

Question 26

How is mRNA stability regulated?

Answer 26

Short-lived mRNAs often have multiple copies of AUUUA sequence in their 3’ UTR (these probably work by stimulating removal of polyA tail)
Length of polyA tail influences stability of mRNA

Question 27

What is RNA splicing?

Answer 27

Carried out by a spliceosome (large ribonucleoprotein complex), which is assembled by interactions of 5 different hnRNP particles (U1-U5)
Spliceosome catalyses two tranesterification reactions that join exons and remove introns which are degraded

Question 28

What is alternative splicing?

Answer 28

A way of increasing the repertoire of proteins that can be made by a single pre-mRNA
Can have optional exons/introns or have internal splice sites (so only part of intron removed)
Means different proteins can be made from same gene

Question 29

How can regulation of mRNA export to cytoplasm regulate gene expression?

Answer 29

Some mRNAs may be functional but selectively degraded in nucleus or exit from nucleus blocked
Could have export blocked by preventing dissociation of spliceosome

Question 30

How can translational control regulate gene expression?

Answer 30

miRNAs bind to complementary 3’ UTR sequences and inhibit translation
siRNAs cause mRNA degradation

Question 31

What is the hierarchy of gene expression control?

Answer 31

Transcriptional initiation
Elongation controls
Capping
Cleavage/polyadenylation
Pre-mRNA splicing
Nuclear export of mRNA
mRNA stability
Translational controls