EXAM 2 - Session 14: Gene Expression Overview

Question 1

Explain what Entinostat is.

Answer 1

Entinostat is a specific inhibitor of class 1 histone deacetylases (HDACs)

Question 2

What does DNA and protein interactio affect?

Answer 2

DNA and protein interaction affects gene expression
* post-translational modifications of chomatin proteins –> control transcriptional access to gene

Question 3

Compare genome versus epigenome.

Answer 3

Genome - DNA sequence
Epigenome - chemical modifications to DNA and associated proteins that don’t modify the DNA sequence (still controls gene activity)
* e.g. histone acetylation

Question 4

Describe histones and how they interact with the DNA backbone.

Answer 4

Histones are small basic proteins that have a positively charges lysine R group.
* positively charged R group interacts with negatively charged DNA phosphate on the backbone.

Question 5

How many types of histones are there? Where are they located?

Answer 5

• 5 types of histones
• 4 types at the histone core
• 1 type as spacer between nucleosomes (caps off each nucleosome)

Question 6

How big are nucleosomes?

Answer 6

~10 nm in diameter

Question 7

How big are chromatin fibers?

Answer 7

~30nm in diameter

Question 8

What affects packing of chromatin fiber?

Answer 8

• histone phosphorylation
• methylation
• acetylation

Question 9

What happens in histone acetylation?

Answer 9

NH3+ group on lysine is covalently modified –> reduces interaction between histones and DNA

Question 10

What are Histone Acetyl Transferases?

Answer 10

HAT’s transfer acetyl groups to lysine side chain of histone.

Question 11

What do HATs cause to happen during histone acetylation?

Answer 11

• Acetyl group is added to lysine
• there is no longer an electrostatic interaction between the DNA phosphate and the histone
• Histones unpack
• DNA transcription is permitted –> gene is expressed

Question 12

What are Histone Deacetylases?

Answer 12

HDACs remove acetyl groups from lysine.

Question 13

What occurs as a result of HDACs?

Answer 13

• HDACs remove acetyl groups from lysine
• the positive charge of lysine interacts with the neg. charge of the DNA phosphate group
• Histones and DNA are able to compact (bc of neutral charge - no repelling)
• transcription is limited –> little gene expression (gene silencing)

Question 14

Explain HDAC inhibitors.

Answer 14

• histone acetyl is not removed
• HATs continue to acetylate histones (add acetyl group)
• “open” chromatin structure
• expression of genes is maintained or initiated from many but not all genes
• more transcription of genes that encode for proteins that inhibit cell growth –> less cell replication

Question 15

What is the TATA box?

Answer 15

Specfic DNA sequence at the start of the gene in the core promoter

Question 16

Describe what promoters are.

Answer 16

DNA upstream of a gene where relevant proteins (such as RNA polymerase and transcription factors) bind to initiate transcription of that gene.

Question 17

What binds to the TATA box/promoter?

Answer 17

transcription factor and RNA polymerase

Question 18

Explain the role of transcription factors.

Answer 18

Aid RNA polymerase interaction with promoter

Question 19

What/Where is the terminator sequence?

Answer 19

Terminator sequence causes release of RNA polymerase downstream of gene.

Question 20

Name the two domains of transcriptional proteins.

Answer 20

DNA-binding domain (DBD) and activation domain

Question 21

Explain the DNA-binding domain (DBD).

Answer 21

DNA-binding domain (DBD) - just binds DNA
* protein-DNA physical interaction through major or minor groove of helix
* MUST be in sequence with DNA-binding domain to stimulate transcription

Question 22

Explain the activation domain.

Answer 22

• stimulates transcription if in sequence with DNA-binding protein
• scaffold to recruit more transcription-enhancing proteins
• often rich in aspartate and glutamate (acidic aa may interact with histone protein)

Question 23

Describe the significance of introns during mRNA processing.

Answer 23

introns - interrupting non-coding sequences
* cut out from mRNA by spliceosomes

Question 24

Describe the significance of exons during mRNA processing.

Answer 24

Expressed coding sequences
* covalent bonds re-established after removal of introns

Question 25

During mRNA processing, what happens to the ends of mRNA?

Answer 25

Modifying both ends of mRNA can slow degradationand increase mRNA half-life.
* modified guanine “cap” added to 5’ end
* poly-A-tail added to 3’ end
* cap and tail are NOT gene coded
* post-transcriptional

Question 26

When do mRNAs leave the nucleus?

Answer 26

mRNA’s are processed before leaving the nucleus
* exported from nucleus AFTER splicing, capping, and tailing is done

Question 27

Describe the mRNA as its final form after exported from nucleus.

Answer 27

mRNA is longer than the amino acid coding info it contains.
* leader and trailer segments are transcribed from DNA
* leader: responsible for mRNA interaction with ribosome subunit
* trailer: AAUAAA signals transcription end and addition of poly A

Question 28

What is SMA?

Answer 28

SMA is the leading genetic cause of mortality in infants <2 years old.
* insufficient muscle innervation –> paralysis and death

Question 29

Explain the protein and gene abnormality that leads to SMA.

Answer 29

• SMN protein is required for nerve-muscle innervation protein from either SMN1 or SMN2 genes
• In SMA, mutated SMN1 genes produce truncated proteins that get degraded –> degrade spinal cord motor neurons
• In SMA, SMN2 genes are normal –> but SMN2 pre-RNA is variably spliced –> splicing removes #7 exon –> resulting protein is easily degraded and cannot compensate for SMN1 protein loss

Question 30

How can mRNA splicing be a therapeutic target for SMA?

Answer 30

Spinraza - anti-sense oligonucleotide serves as splicing guide of the pre-mRNA
* #7 exon is retained
* Stable, full-length SMN2 protein is produced
* if there is enough produced, it can support nerve-muscle innervation.