Eukaryotic transcription

Question 1

general vs specific TFs

Answer 1

GENERAL: bind to promoter (eg. TATA box) and RNAP and allows it to initiate transcription

SPECIFIC: bind to enhancer or silencer regions regions to either stimulate or inhibit rate of transcription

Question 2

3 classes of RNAPs

Answer 2

RNAP1: codes for rRNA

RNAP2: codes for mRNA, miRNA and snRNA

RNAP3: codes for tRNA and small amounts of rRNA

Question 3

2 parts of eukaryotic promoters

Answer 3

includes ALL sequences important to control transcription of the gene hence made of 2 parts:
1. CORE PROMOTER
2. UPSTREAM REGULATORY ELEMENTS

Question 4

Ways that eukaryotes modulate extent of gene regulation

Answer 4

REGULATORY SEQUENCES:
1. ENHANCERS: increase transcription rate

2. SILENCER: decrease transcription rate

Question 5

how do enhancers/silencers work

Answer 5

enhancer/silencer region a certain distance away from promoter is bound to a specific TF. This causes a conformational change so the DNA loops. E/S region is brought close to promoter (close proximity) and hence the specific TF interacts with general TF & RNAP bound to promoter to either stimulate or inhibit transcription rate

Question 6

Structure of eukaryotic core promoters

Answer 6

upstream promoter boxes:
TATA box (dominating) at -25
BRE

Inr (initiator)at +1

Downstream promoter box:
DPE

Question 7

Initiation process

Answer 7

-general TF2 sequence of binding to form preinitiation complex
-formation of transcription bubble
-CTD phosphorylation to trigger disengagement of RNAP2 from promoter (conformational change causing tighter interactions between RNAP2 and DNA which helps in elongation)
-Clearance of promoter after passing into elongation

Question 8

Elongation process

Answer 8

-presence of elongation factors to: suppress pausing of RNAP2, prevent arrest of enzyme and modify chromatin to aid elongation
-reading of template strand by the enzyme to add complimentary rNTPs

Question 9

Termination process

Answer 9

-termination signal is the RNAP2 reaching the polyadenylation sequence (AAUAAA)
-Cleavage and polyadenylation specificity factor (CPSF) found on RNAP2 is associated to the polyadenylation sequence
-this activates certain enzymes to cleave RNA and releases it.

Question 10

General transcription factors (6)

Answer 10

TFII: A,B,D,E,F,H and MPC (mediator protein complex)

Question 11

TBP/TF2D conversion

Answer 11

TBP+ 11TAFs = TF2D

TAF: tbp associated factors (coactivators)

Question 12

Detailed general TF binding sequence required for RNAP2 promoter binding

Answer 12

1. TF2D binds strongly to TATA box (or other promoter box present)
2. TF2D allows binding of TF2A and then TF2B which interacts with DNA and TBP after it associates. This links TF2B to initiation complex
3. Association of TF2F with RNAP2 which allows binding of enzyme to pre-initiation complex (closed)
4. Binding of TF2E and then TF2H which act as helicases to open DNA strand and covert closed complex to open complex
5. TF2H also phosphorylates Ser5 on CTD RNAP2 tail to allow passage into elongation
6. PROMOTER CLEARANCE: all TFs other than TBP are released from promoter so the process can restart if necessary

Question 13

Structure and role of TF2H

Answer 13

Serine threonine kinase with 9 subunits: phosphorylates Serine (5) residues on CTD RNAP2 tail to allow passage into elongation

Question 14

CTD tail structure

Answer 14

CTD: carboxy terminal domain portion of RNAP2 tail. Repeated sequence of 7 amino acid residues

unphosphorylated: participating in the formation of the initiation complex

phosphorylated (by TF2H): RNAP can move fast and pass into elongation

Question 15

role of MPC

Answer 15

MPC = mediator protein complex
binds to UNPHOSPHORYLATED RNAP (during initiation) and acts as a molecular bridge (co-activator)

links RNAP complex and other activator/repressor molecules that regulate rate of transcription

Question 16

coactivator def

Answer 16

a protein that aggregates/ coagulates for activation or repression but DOESNT Bind to dna

Question 17

consensus sequence def

Answer 17

contains a breakdown of the most probable sequence of nucleotides

Question 18

difference between enhancers and regulatory elements

Answer 18

enhancers can affect transcription independent of distance away from the promoter, however a proximal regulatory element must be positioned close to the promoter to have an effect (distance dependent)

Question 19

Structure of TFs

Answer 19

Made of structural and functional domains:

1. DNA binding domain (identify cis elements)
2. Flexible protein domains (linking domains 1 and 3)
3. trans activation domain (final domain that activates transcription)

Question 20

How are DNA binding domains of TFs categorised?

Answer 20

they dont have to have the same sequence but can be grouped into families depending on common secondary structures

Question 21

structure and function of the helix-turn-helix domain

Answer 21

-DNA BINDING DOMAIN present in both TFs of euk and pro cells

Contains 3 alpha helix domains:
-helixes 1 and 2 are parallel
-helix 3 is perpendicular to 1&2
connected by loose DNA with no 2ary structure

FUNCTION:
-helix 1/2 bind to DNA without recognizing specific sequences (only help the TF to come into close proximity with the DNA)
-helix 3 inserts itself into DNA groove and recognizes consensus sequence to bind to

Question 22

structure and function of the zinc finger domain

Answer 22

DNA BINDING DOMAIN present in large amounts

C2H2 zinc-finger proteins: 2 cysteines connecting to 2 histidines via association of a central Zn ion. This formed an alpha helix connected by the Zn ion to 2 beta pleated sheets

FUNCTION:
a helix recognises the specific DNA binding sequence
beta sheets maintain structure and allows some interaction with backbone of the DNA

!!each zinc finger can recognise 3 nucleotides

Question 23

are TFs present in low or high numbers in our cells

Answer 23

low numbers because if you increase the concentration of TF, the probability of off target transcription (recognition of the wrong genes by the TFs) would increase and lead to errors

Question 24

how are TF trans activation domains classified

Answer 24

they don’t have a specific sequence of amino acids or secondary structures

hence they are classified into 3 classes based on abundance of:
1. -ve charged amino acids
2. glutamine
3. proline

Question 25

mode of action of TF activation domains

Answer 25

Involves their property to adapt their structure depending on the surface they want to interact with (HENCE take on certain secondary structures when coming into contact with certain charges)