L14: Transcriptional Regulation in Eukaryotes

Question 1

transcriptional regulation in eukaryotes vs prokaryotes

Answer 1

1. eukaryotes have nucleosomes (comprised of histones)
2. eukaryotic genes have more regulatory sequences and more transcriptional regulators

Question 2

transcriptional regulation in eukaryotes vs prokaryotes - nucleosomes

Answer 2

• the genes are partially concealed from being expressed
• this creates an obstacle but also adds a new level of gene regulation

Question 3

transcriptional regulation in eukaryotes vs prokaryotes: regulatory sequences and transcriptional regulators - promoter and regulator binding sites

Answer 3

• promoter (in prokaryotes): region to which transcriptional machinery binds
• regulator binding sites (in eukaryotes): DNA recognition sites bound by transcription factors

Question 4

transcriptional regulation in eukaryotes vs prokaryotes - what makes regulatory binding sites so different than promoters?

Answer 4

• they are more in number and can be positioned at a large distance from the gene
• it allows greater integration of signals for a gene
• it is bound by activators and repressors

Question 5

activator proteins - yeast transcriptional activator Gal4

Answer 5

• Gal4 dimerizes and activates galactose metabolism genes (GAL4)
• it binds to a regulatory sequence called UAS (upstream activating sequence)
• consists of 2 modular and separable domains

Question 6

activator proteins: Gal4 - what are the 2 modular and separable domains?

Answer 6

1. DNA-binding domain: recognizes UAS
2. Activation domain: recruits transcriptional machinery

Question 7

DNA-binding domains - eukaryotic transcriptional regulators

Answer 7

• often inserts an alpha-helix into the major groove of DNA
• termed recognition helix

Question 8

DNA-binding domains example

Answer 8

• there are many classes
• Ex: Zinc-finger domain

Question 9

DNA-binding domain example - Zinc-finger domain

Answer 9

• incorporates zinc atoms into structure
• alpha-helix inserted into the major groove

Question 10

how can activators initiate transcription?

Answer 10

1. recruitment of RNA pol II indirectly (prokaryotes do so directly)
2. recruitment of nucleosome modifiers

Question 11

how can activators initiate transcription - RNA Pol II

Answer 11

• interaction is through complexes such as Mediator or TFII subunits
• mediator bridges activators with RNA Pol II

Question 12

how can activators initiate transcription - nucleosome modifiers

Answer 12

1. chemically modify histones (Histone acetyltransferases of HATs) where histone acetylation loosens the chromatin to reveal DNA-binding sites
2. displace or “remodel” nucleosomes (chromatin remodeling complex)

Question 13

what are enhancers?

Answer 13

• cis-regulatory elements bound by regulatory transcription factors
• they dictate differential gene expression
• only cells that have the regulatory protein will express the gene

Question 14

what are the characteristics of enhancers

Answer 14

1. they can be 100,000 bases from the promoter and can be found in introns upstream or downstream of genes
2. most genes have more than one enhancer
3. enhancers can still function if moved or flipped in 5’ to 3’ orientation

Question 15

explain the control of transcriptional regulator activity

Answer 15

• a signal input can alter the the activity of transcriptional regulators
• ex: unmasking of an activating region

Question 16

control of transcriptional regulator activity - unmasking of an activating region

Answer 16

• Gal4 activates the GAL1 gene only in the presence of galactose (and in the absence of glucose)
• this is because galactose triggers the release of the masking protein Gal80 from Gal4

Question 17

how are activators in eukaryotes different compared to prokaryotes

Answer 17

• multiple activators often work together synergistically by cooperative binding
• the DNA binding of one protein aids or requires the binding of a second

Question 18

activator mechanisms in eukaryotes

Answer 18

1. direct interaction
2. indirectly through a common third protein
3. indirectly through recruitment of a nucleosome remodeler

Question 19

activator mechanisms in eukaryotes - directly

Answer 19

two activators (A and B) move to binding site and touch each other

Question 20

activator mechanisms in eukaryotes - indirectly via a third protein

Answer 20

two activators (A and B) move to binding site but they can only interact with each other if protein X gets in between them

Question 21

activator mechanisms in eukaryotes - indirectly via nucleosome remodeler

Answer 21

• one molecule (A) is bound properly but B is not due to histone wrapping DNA too tightly
• A then recruits a modifier than then causes unwinding so B can bind

Question 22

why is the regulation of activators important for signal integration?

Answer 22

• the gene is only activated when two signals are received
• each signal is communicated by a separate activator

Question 23

signal integration at HO locus in yeast

Answer 23

• the HO gene is only expressed in the mother cell at G1-S transition of the cell cycle
• it is regulated by 2 factors: SWI5 and SBF

Question 24

signal integration at HO locus in yeast - SWI5

Answer 24

• it is only active in the mother cell and communicates its identity
• the binding site is too distant to activate HO expression
• it recruits chromatin modifiers that act on the SBF binding site

Question 25

signal integration at *HO* locus in yeast - SBF

Answer 25

- it is only present in the bud - it communicates the timing of G1-S transition - it requires chromatin remodeling in order to bind near *HO* - SBF binds on DNA and recruits Mediator to activate *HO* expression

Question 26

how are repressors different in eukaryotes vs prokaryotes

Answer 26

eukaryotic repressors do not bind to DNA sites overlapping the promoter to block RNA Pol binding

Question 27

repressor mechanisms

Answer 27

1. competition 2. inhibition 3. indirect repression

Question 28

repressor mechanisms - competition

Answer 28

the repressor binds to the region that overlaps with an activator binding site

Question 29

repressor mechanisms - inhibition

Answer 29

the repressor binds near the activator and shields its activating region

Question 30

repressor mechanisms - indirect repression

Answer 30

the repressor recruits a histone modifier that alters nucleosomes to inhibit transcription

Question 31

indirect repression example

Answer 31

- ***GAL1* gene** - its involved in galactose metabolism (activated by Gal4) - the factor **Mig1** binds upstream of *Gal1* in the presence of glucose - Mig1 recruits **Tup1** repression complex (contains HDAC)

Question 32

*GAL1* gene - what happens when only glucose is present?

Answer 32

- **bc glucose is present**: the Tup1 repression complex is bound to DNA - **bc galactose is not present**: Gal4 is repressed by Gal80 bc it sits on top of its activation domain - results in **no** GAL1 expression

Question 33

*GAL1* gene - what happens when galactose and glucose is present?

Answer 33

- **bc galactose is present**: Gal80 unbinds and allows the activation domain of Gal4 to be exposed - **bc glucose is present**: Tup1 repression complex is bound to DNA - results in **no** expression of GAL1

Question 34

*GAL1* gene - what happens when only galactose is present?

Answer 34

- the Tup1 repression one complex is not bound to DNA - Gal80 disassociates from Gal4 allowing the activation domain to be accessible - results in GAL1 expression

Question 35

how is the *GAL1* gene in eukaryotes similar to the Lac operon in prokaryotes?

Answer 35

two carbohydrate inputs control gene expression