Lecture 10 - Modular mechanisms of transcription activation and repression

Question 1

what is a zinc-figer motif

Answer 1

protein regions that fold around a central Zn2+ ion.

Question 2

2 zinc finger motifs and their occurence

Answer 2

C2H2 zinc finger –> most common motif in humans

C4 zinc finger –> found in approx. 50 human TFs

Question 3

What are C and H in C2H2 and C4 zinc fingers

Answer 3

a.a cystine and histidine

Question 4

C2H2 zinc finger how it works, how many of this motif the domain contains, how domain/prot. bind

Answer 4

Binding of 2 C and 2 H to zinc compacts the domain and allows insertion of alpha helix in the major groove.
Usually domain contains 3 or more C2H2 motifs
Motifs bind as monomers

Question 5

C4 zinc finger how many of this motif the domain contains, how domain/prot binds.

Answer 5

Domain contains only two motifs

Binds as homodimers. Note : like C2H2, fingers insert in DNA

Question 6

Something particular about binding of domains that have C4 zinc finger motif

Answer 6

Domains have two-fold rotational symmetry and bind to consensus DNA sequences that are inverted repeats.

Question 7

2 other DNA binding domains

Answer 7

leucine zipper. basic helix-loop-helix (bHLH)

Question 8

Leucine zipper motif. Leucine characteristic. and consequence

Answer 8

Leucine residue at every seventh position. Leucine is hydrophobic so goes inwards and gives helical form to helices of zipper

Question 9

How leucine zipper binds DNA

Answer 9

as dimers, often heterodimers

Question 10

Proteins like leucine zipper principle and how you call them

Answer 10

'’Basic zipper (bZip)’’ is the name of the larger family. Prots in this family have a zipper with a different hydrophobic a.a at every seventh position

Question 11

basic helix-loop-helix description (diff. w/ basic zipper)

Answer 11

similar to basic zipper but a nonhelical loop seperates two alpha-helical regions.

Question 12

How bHLH containing proteins bind

Answer 12

Bind as heterodimers

Question 13

Activation domain a.a usually found

Answer 13

usually high percentage of one or two a.a (Asp, Glu, Gln, Pro, Ser, Thr)

Question 14

Acidic activation domain what it’s made of and something particular

Answer 14

high percentage of Asp or Glu and is active when bound by a coactivator

Question 15

Ex. of acidic activation domains (2)

Answer 15

1) RARgamma has to bind retinoic acid to be in an active conformation
2) CREB must be phosphorylated to bind its coactivator CBP and become an active TF.

Question 16

Activation domains before they bind co-activator

Answer 16

may be random coils/unstructured

Question 17

Other exemple of act. domain w/ coactivator and how it works

Answer 17

Estrogen receptor ligand-binding activation domain
When estrogen (grey) binds activation domain (brown), a green alpha helix interacts with the ligand (domain -> brown). Alpha helix of domain bends and can bind alpha helix subunit of another coactivator

Question 18

Repressor of estrogen binding in estrogen receptor ligand binding activation domain

Answer 18

Tamoxifen. Blocks binding of estrogen. Domain can’t become active

Question 19

Length of repression domains (and activation domains)

Answer 19

may be short (<15 a.a.)

Question 20

Repression domains, 2 (obvious) things it does

Answer 20

Binds co-repressor proteins and mediates protein-protein interaction.

Question 21

Repressors and activators DNA binding in common (2 things to note)

Answer 21

1) Certain repressors can block activators by binding to their DNA sequence
2) Repressors and activators can bind to common sequences or have their specific sequences

Question 22

Exemple of repressor and how it interacts with DNA

Answer 22

Bacteriophage 434. Helix interacts with MAJOR groove

Question 23

Why are TFs required

Answer 23

basal transcription is very low

Question 24

3 things that can generate transcription factor regulatory diversity

Answer 24

Heterodimers, Inhibitory factors, Cooperative binding of unrelated TFs to nearby sites

Question 25

Heterodimers : how they can generate TF regulatory diversity

Answer 25

In some heterodimers, each monomer has different DNA-binding and activation/repression domain specificity. Therefore, different combinations between monomers can create diversity.

Question 26

Inhibitory factors how they work (what 2 known motifs they act on specifically)

Answer 26

block DNA binding by some bZip and bHLH monomers, (which bind as heterodimers)

Question 27

What is necessary if you want to use inhibitory factors to create TF regulatory diversity ?

Answer 27

You have to express the inhibitory factors

Question 28

How cooperative binding of unrelated TFs works

Answer 28

If they bind alone to DNA, their bining is weak. They bind together and then bind to DNA and their binding to DNA becomes strong

Question 29

Why cooperative binding of unrelated TFs enhances their binding to DNA

Answer 29

prot-prot interaction stabilizes their binding

Question 30

Exemple of 2 TFs that do cooperative binding

Answer 30

NFAT and AP1

Question 31

What links activation domain of TFs to RNAP II at the promoter and how

Answer 31

mediator complex -> forms molecular bridge between activation domain and Pol II

Question 32

What could we consider the mediator complex to be

Answer 32

A co-activator of transcription

Question 33

How does the mediator complex acting as a bridge between activation domain and Pol II activate transcription

Answer 33

Activation-domain + mediator interactions STIMULATE assembly of pre-initiation complex at promoter.

Question 34

Which part of mediator complex interact with Pol II and how and what other parts of mediator may interact with ?

Answer 34

Head and middle of mediator complex interact directly with RNAP II SUBUNITS. Other mediator domains interact w/ activation domains of activators that are bound to ENHANCERS OR PPEs

Question 35

Steps of eukaryotic transcription initiation control

Answer 35

1) Pioneer TF binds to sequence in condensed chromatin and recruits chromatin modelling complexes
2) Chromatin is open so activators bind enhancers and PPEs interact with each other and with subunits of mediator complex to assemble preinitiation complex (recruit Pol II and GTFs)

Question 36

How eukaryotic transcription activators/repressors affect gene expression (2 ways)

Answer 36

1) Interact with co-activators/co-repressors that modulate chromatin structure
2) Interact with Pol II and GTFs

Question 37

What is required for assembly of PIC in vivo and how cell manages that

Answer 37

several activators. Cell produces specific set of activators required for transcription of a particular gene to express it.