Lecture 38: Eukaryotic Gene Regulation

Question 1

3 classes of transcirption factors

Answer 1

1) DNA Binding Proteins
2) mediator proteins
3) chromatin remodeling enzymes

Question 2

Epigenetic changes

Answer 2

alter gene expression by changing access of transcription factors to DNA sequences

histone modification
DNA Methylation

Question 3

HATS (histone acetyl transferases)

Answer 3

faciliate gene activation

Question 4

HDACs (histone deactylases)

Answer 4

inhibit gene expression

Question 5

steroid receptors

Answer 5

function as ligand dependent and independent regulaturs that recruit HATs and HDACs

Question 6

why remodel nucleosomes

Answer 6

they can block DNA binding to specific sequences

remodel in orer to premit gene expression

Question 7

what is a nucleosome?

Answer 7

protein core of histones with DNA wrapped around it

Question 8

unpacking chromatin structures

Answer 8

use HAT enzymes to add acetyl groups

open up the nucleosomal structures

Question 9

repackaging DNA into chromatins

Answer 9

remove acetyl groups with HDAC

Question 10

if chromatin condensed, chromatin

Answer 10

OFF

deacetylated

Question 11

if chromatin decondensed, chromatin

Answer 11

ON

acetylated

Question 12

epigenetics is what

Answer 12

chromatin modifications that alter transcription factor binding

acetylation or deacytlation by HATs and DHACs

Question 13

what is the acetyl group donor in the HAT reaction

Answer 13

coenzyme A

Question 14

what is the methyl group donor in the DNMT reaction

Answer 14

S-adenosylmethionine

Question 15

How does acetylation change the structure of chromatin

Answer 15

pos charged lysine, acetylation takes away this pos charge, now its neutral

INTERACTS LESS WELL with NEG DNA since no longer pos charged

Question 16

methylation in DNMT… how does this change the structure of DNA?

Answer 16

add methyl group to histone tail (when txn is ON)

to tigthen up and turn TXN OFF

Question 17

Binding of chromatin remodeling complex (CRC) to tightly wound DNA

Answer 17

shifts postions of nucelosomes to make DNA binding sites accessible

Question 18

3 types of sequence specific binding proteisn that chromatin remodeling uses

Answer 18

1)Basal transcption factors (likeTATA binding protein (TPB_)
not super specfic. bind to promoter to recruit Pol II
2) transcriptional actibator proteins
bind to sequence near promotorer and recruit HAT and CRC
3) transctiption repressor proteins
recruit HDAC and cytosine metholtransferase enzymes to repackage chromatin

Question 19

TBP, a sequence speciic DNA binding protein

Answer 19

works at major groove,

recruits LOTS of other proteins to form complexes in order to get polymerase to right place

Question 20

recognizing DNA sequences… helical elements

Answer 20

some side chains are really good at recognizing H bond patterns in DNA
mostly through interactions of heliciles in Major grove

Question 21

3 protein structural motifs that insert helical elements into major groove

Answer 21

leucine zipper motif
zinc finger motif
homdeodomain motif

Question 22

leucine zipper motif

Answer 22

has lots of leucines repeats for hydrophobic interactions that drive dimerization of 2 helicies

2 helicies (1 in front and 1 in back) that are inserted into major groove of DNA

part of larger protein complex

Question 23

Do DNA binding proteins bind covalently or noncovalently to DNA? explain

Answer 23

noncovalently

easier to remove them, reversible, can go on or off depending on needs.

Question 24

zinc finger motif

Answer 24

helix and loop are stablized by zinc ions
zinc finger are used to design DNA binding proteins that string the zinc fingers together so that you can target the sequence that you want

TAA USUALLY. Ts and As are important

Question 25

homdeodomain motif

Answer 25

part that contacts DNA is small, makes contracts in major groove

Question 26

specificity… finding what sequence a zinc finger prefers to bind to

Answer 26

give it a piece of DNA, randomize the sequence
see if it binds to it or not
free DNA smaller than bound DNA

Question 27

gel shift assays

Answer 27

if theres a band, they say proteins will bind to that particular sequence

Question 28

investigating comonallities of the binding

Answer 28

see that core has all Ts and As

some variability around that as you go further from the core

Question 29

the core

Answer 29

what is found in every single position

Question 30

enhancer sequences

Answer 30

bound by activator protein
can be near or far from TATA box

after bound by HAT,
recruit the other proteins,
mediator interacts with TBP and other transcription factors
TXN starts

Question 31

where are enhancers

Answer 31

they can be far from start site because of looping out, they are eventually brought close to each other

Question 32

Activator–> Mediator–>TBP–>???

Answer 32

RNA Pol

Question 33

so what is bound

Answer 33

activator, mediator, TBP, recrut more transcription factors and RNA Pol 2, CTD stuff (poly A, splicesomes, capping stuff)
TRANSCRIPTION HAPPENS

Question 34

Tissue specific Gene expression… how does this occur

Answer 34

by the state of chromatin packaging and presence/abseences of transcritption factors in different tussues

Question 35

when are acetylation and cytosine methylation patterns established>

Answer 35

Early in development

affects expression of transcription factors in different cell types

Question 36

“master” regulators

Answer 36

control expression of functionally related genes in tissues

Question 37

Steroid receptors

Answer 37

great exampmple of eukaryotic gene regulation
part of nuclear receptor family
gluccocorticoid receptr
estrogen receptor
PPAR proteins
retinoid receptors
vitamin D receptors

Question 38

what do steroid receptor proteins contain?

Answer 38

zinc finger DNA binding motif linked to binding domain with hydrophobic pocket

Question 39

what are steriod receptors (activators or repressors)

Answer 39

both gene activators and repressors

depends on chem properties of the ligands and the gene sequence they bind to

Question 40

what residues are important for binding to the zinc that stablizes the zinc finger structure?

Answer 40

cystine

Question 41

FOUR models of gene regulation by nuclear receptors

Answer 41

1) Ligand-dependent direct activation
2) Ligand-dependent direct repression
3) Ligand-independent direct repression
4) Ligand-dependent indirect repression

Question 42

Ligand-dependent direct activation

Answer 42

when nuclear receptors (dimers) are in presence of their ligand, they bind to DNA b4 start site
bind to gene and ligand results in recruiting HAT
HAT decondeses chromatin, TXN turned on

Question 43

Ligand-dependent direct repression

Answer 43

nuclear receptor is associated with protein
ligand binding recruits HDAC
gene expression blocked

Question 44

Ligand-independent direct repression

Answer 44

nuclear receptors are already bound to DNA
other factors don’t require direct binding of ligand to receptor, causes recruitment HDAC
blocked gene
RARE!!!!!

Question 45

Ligand-dependent indirect repression

Answer 45

Ligand binds to nuclear receptor
Receptor does not have direct DNA binding, blocks gene expression in another way (perhaps interaction with other transcription factors)

Question 46

What is the purpose of the nuclear receptor regulation?

Answer 46

whether HDATS or HATs will be recruited to turn off or on

Question 47

What are mechanistic differences between prokaryotic/eukaryotic TXN factors?

Answer 47

EU: mostly by enzyme activity (HAT and HDAT). recruit enzymes for modification of chromatin
EU: interact more indirectly, don’t directly interact with RNA Pol, action at a distance
PRO: all about binding