Lecture 7: Eukaryotic Gene Expression Transcription

Question 1

What are the differences between eukaryotic and prokaryotic gene expression?

Answer 1

Prokaryotic vs Eukaryotic
polycistronic monocistronic
cytoplasmic nuclear
coupled transc-transl not coupled
single RNA polym. 3 diff RNA polym
uninterrupted interrupted (splicing)

Question 2

What does polycistronic mean?

Answer 2

more than 1 proteins coded for in one mRNA transcript

Question 3

what do the three RNA pol. transcribe?

Answer 3

1. rRNA
2. mRNA (via hnRNA)
3. tRNA

Question 4

What are the three factors that can interact with RNA pol2 to modify transcription?

Answer 4

1. GENERAL FACTORS: form basal transcription apparatus with RNA pol2 itself
2. UPSTREAM FACTORS: DNA-binding, recognising short sequences, unregulated
3. INDUCIBLE FACTORS: recognised to control gene expression in a stimulus specific manor

Question 5

What is the core promoter?

Answer 5

‘minimal DNA elements required for accurate transcription initiation by RNA polymerase 2’

Question 6

What are the accessory factors of RNAP2? How many are there and what are they?

Answer 6

6 general initiation factors; TF2D, TF2A, TF2B, TF2F, TF2E, TF2H

RNAP2 + these factors = basal transcription apparatus

Question 7

What is the pre-initiation complex (PIC)?

Answer 7

“The preinitiation complex is a complex of approximately 100 proteins that is necessary for the transcription of protein-coding genes in eukaryotes and archaea”

Question 8

What is TF2D composed of? What is its purpose?

Answer 8

Assembly of a pre-initiation complex (PIC)

Promoter recognition:

• TF2D = TBP + TAFs
• TBP (TATA-binding protein)
• TAFs (TBP-associated factors)

This is used to position RNAP2 in the right place. Also binds DNA in minor groove (binds DNA ~80 degrees)

Question 9

What is the role of TF2D (TBP + TAFs) in PIC assembly?

Answer 9

1. recognise core promoter (TATA box)

2. Recognise core promoter (no TATA box)

Question 10

What is the role of TF2A in PIC assembly?

Answer 10

Stabilise TBP and TAF binding

Question 11

What is the role of TF2B in PIC assembly?

Answer 11

Aids RNAP2/TF2F recruitment and transcription start site selection

Question 12

What is the role of TF2F in PIC assembly?

Answer 12

1. Brings RNAP2 to promoter

2. Breaks non-specific RNAP2-DNA interactions

Question 13

What is the role of TF2E in PIC assembly?

Answer 13

1. Recruits TF2H
2. Regulates TF2H helicase
3. ATPase and kinase activities

Question 14

What is the role of TF2H in PIC assembly?

Answer 14

1. Aids promoter melting (dsDNA to ssDNA)

2. phosphorylation of CTD for promoter clearance

Question 15

How many repeats are there in RNAP2 C-terminal domain (CTD)? What amino acids are subject to phosphorylation?

Answer 15

52 repeats of YSPTSPS in mammals

Ser2 and Ser5 subject to phosphorylation

Question 16

What is unphosphorylated RNAP2A used for

Answer 16

Initiation. Interacts with general initiation factors.

Question 17

What is phosphorylated RNAP2O used for?

Answer 17

Elongation.

1. TF2H (and others) is CTD kinase
2. allows dissociation of first TF2B and E then TF2H
3. TF2F remains associated

Question 18

What are the roles of RNAP2 CTD?

Answer 18

1. Splicing
2. Capping
3. Addition of polyA tail

Question 19

How is transcription activated and repressed?

Answer 19

1. Cis-acting control elements (DNA)
   a. promoters
   b. enhancers/repressors
2. Trans-acting transcription factors
   (protein)
   a. constitutively expressed or induced
   b. two domain (DNA binding and
   activation domains)

Question 20

Where is TATA box located? Where is the CAAT box located? Where is the GC box located?

What happens as a result of mutations in these regions?

Answer 20

TATA box = -30
CAAT box = -75
GC box = -90

Mutation results in far lower transcription levels

Question 21

How do promoters ‘mix and match’

Answer 21

They contain different numbers of TATA, CAAT, GC boxes, and other elements

“TRANSCRIPTION OF GENE A CONTROLLED BY ACTIVATORS 1, 2, 3, and 4. TRANSCRIPTION OF GENE B CONTROLLED BY ACTIVATORS 2, 4, and 6”

Question 22

How can a combination of control elements activate transcription?

Answer 22

Only when activator proteins are present (and potentially activated)

Question 23

What are the two domains of TFs?

Answer 23

1. DNA-binding domain
   
   • recognise target sequence in enhancers
     or promoters
2. Activation domain
   
   • function to influence transcription by
     contacting basal apparatus

Question 24

What are the protein structure motifs in DNA binding?

Answer 24

1. Helix-turn-helix (e.g., homeodomain 
    containing proteins)
2. Zinc finger (e.g., steroid receptors)
3. Helix-loop-helix (e.g., c-Myc)
4. Basic region-leucine zipper                
    (e.g., Fos/Jun = AP1

Question 25

What is a transcriptional Co-activator?

Answer 25

“Does not bind to DNA itself, but bridges between the activator and the basal transcriptional apparatus”

Question 26

What does CRE, CREB, and CBP mean?

Answer 26

1. CRE = cyclic AMP response element
2. CREB = CRE binding protein
3. CBP = CREB binding protein

Question 27

What is an enhancer?

Answer 27

They are “distance, orientation and position independent” from the promoter

Question 28

What is transient transfection?

Answer 28

1. Obtain genomic clone of gene - obtain 
    'promoter region'
2. Obtain reporter vector e.g., 
   a. CAT
   b. Luciferase
   c. beta-gal
   d. GFP
   - sub clone promoter fragment 
3. Transfect into cells
   a. CaPO4
   b. DEAE-dextran
   c. Lipid
   d. Electroporation
   - measure reporter under test conditions