Part 3: Eukaryotic Regulation of transcription

Question 1

How do non-steroid hormones activate transcription of genes?

Answer 1

• by activating cell-surface receptors, which in turn activate intracellular signal transduction pathways

Question 2

How do steroid hormones activate the transcription of genes?

Answer 2

• by directly stimulating gene transcription as hormone-receptor complexes.

Question 3

Class II genes are:

Answer 3

• protein-encoding genes
• transcribed by RNA Pol II in mRNA

Question 4

Chromatin:

Answer 4

• complex of DNA plus associated proteins

Question 5

Steroid hormone receptors:

Answer 5

• soluble proteins
• act as transcriptional activators in response to hormone (steroid) binding

Question 6

What other complex does a DNA-hormone-receptor complex interact with to stimulate the RNA pol II transcriptional machinery?

Answer 6

a “coactivator” complex

Question 7

The two functional domains of all steroid hormone receptors:

Answer 7

1. hormone binding domain
2. DNA binding domain
   
   • recognizes specific enhancer elements

Question 8

Upon hormone binding, steroid receptors typically undergo a conformational change to create a:

Answer 8

• transcriptional activation domain
  
  • binds a “coactivator” complex

Question 9

Steroid response elements (SREs):

Answer 9

• DNA sequences that are enhancers for steroid responsive genes
• bind the hormone-receptor complexes, resulting in stimulation of gene expression
• typically palindromic, about 17bp

Question 10

Agonists:

Answer 10

• bind hormone receptors
• stimulate receptor activity and gene expression

Question 11

Antagonists:

Answer 11

• bind hormone receptors
• block receptor activity and represses gene expression

Question 12

The three critical domains of all steroid hormone receptors (in general):

Answer 12

1. ligand-binding domain
2. activator domain
3. DNA-binding domain

Question 13

What does the DNA-binding domain of a steroid hormone receptor bind to?

Answer 13

• an enhancer element
  
  • the steroid response elements (SREs)

Question 14

Steroid response elements (SREs) are specific for:

Answer 14

• specific steroid hormone receptor - complexes
• SREs typically only differ by about 4bp
  
  • therefore, mutations in SREs can bind different steroids, causing wrong gene expression

Question 15

How far away are SREs from the promoter region of a gene?

Answer 15

• far, sometimes thousands of bps away from the promoter

Question 16

Steps in the mechanism of transcriptional activation by steroid hormones:

Answer 16

1. a hormone binds to the ligand-binding domain of a steroid hormone receptor
2. the hormone-receptor complex binds to the SRE (an enhancer) via the DNA-binding domain of the receptor
3. a transcriptional coactivator complex binds to the activation domain of the receptor that in turn is able to stimulation transcription

Question 17

Two ways coactivators stimulate transcription:

Answer 17

1. recruiting or stabilizing RNA pol II/GTF to the core promoter
2. “clearing a path” for RNA pol II by altering the chromatin DNA template (chromatin remodeling)

Question 18

Chromatin is made up of repeating units of:

Answer 18

nucleosomes

Question 19

Each nucleosome contains:

Answer 19

• about 200 bp of DNA
• two copies each of histones H2A, H2B, H3 and H4 (“histone octamer”)

Question 20

Steps by which steroid hormone receptors ultimately activate gene expression by altering chromatin structure:

Answer 20

1. hormone binds to steroid hormone receptor
2. a sequence-specific enhancer element (SRE) binds the hormone receptor-hormone complex
3. a coactivator complex binds to displace or disrupt nucleosomes, thereby clearing a path for the binding of RNA polymerase II and its general transcription factors (TFIIA, TFIIB, etc.)

Question 21

What proteins serve as chromatin remodeling proteins?

Answer 21

certain classes of coactivators

Question 22

The two classes of chromatin-remodeling coactivators:

Answer 22

1. catalyzes covalent modification of histone tails
2. ATP-dependent remodeling complexes
   
   • use the energy of ATP hydrolysis to remodel nucleosomes; do not covalently modify histones

Question 23

What enzymes catalyze the acetylation of histone tails?

Answer 23

histone acetyltransferases (HATs)

(reversible with HDAC)

Question 24

What enzymes catalyze the deacetylation of histone tails?

Answer 24

histone deacetylases (HDACs)

(reversible with HAT)

Question 25

Most covalent modification during chromatin remodeling by coactivator complexes occur on what part of histones?

Answer 25

N-terminal tails

Question 26

What types of covalent modifications can coactivator complexes make to histones during chromatin remodeling?

Answer 26

UMAP

1. ubiquitination
2. methylation
3. acetylation
4. phosphorylation

Mostly on N-terminal tails of the histones

Question 27

Final result of chromatin remodeling:

Answer 27

• promoter region is taken out of the nucleosome loop and exposed so that RNA polymerase can bind to it

Question 28

Five classes of steroid hormones:

Answer 28

PGAME

1. PROGESTINS
2. GLUCOCORTICOIDS
3. ANDROGENS
4. MINERALOCORTICOIDS
5. ESTROGENS

• all cholesterol derivative
• each binds specific hormone receptors to activate different sets of genes

Question 29

Anabolic steroids:

Answer 29

• agonists (trigger signalling pathways)
• bind to the androgen receptor to stimulate expression of genes involved in muscle development

Question 30

Tamoxifen:

Answer 30

• antagonist
• acts as competitive inhibitor to estrogen estradiol
• binds to estrogen receptors, but does not stimulate signalling pathways (disables the receptor)
  
  • fails to induce conformational change necessary for coactivator binding

Question 31

Alpha-amanitin:

Answer 31

• toxin
• analagous to rifampicin
• Binds to RNA polymerase II in humans
  
  • inhibits elongation stage of transcription

Question 32

TFIIH:

Answer 32

• multi-subunit complex
• required for transcription initiation - unwinds so helices can start its job
• Mutations in two subunits of TFIIH cause XP, TTD, and CS - all a consequence of TC-NER inhibition