Lecture 26 - Transcriptional Controls, Control of Gene Expression 1:

Question 1

What is required for transcriptional control of gene regulation?

L26 S10

Answer 1

• short sequences of DNA which act as recognition sites for proteins
• gene regulatory proteins

Question 2

What part of DNA do regulatory proteins bind?

L26 S12

Answer 2

-major groove

Question 3

What modules will be present on all DNA transcription factors?
What modules may be present but not always?

L26 S16

Answer 3

Always present:

• DNA-binding module
• activation module

Sometimes present:

• dimerization module
• regulatory module

Question 4

What are the different types of DNA-binding motif structures?

L26 S21

Answer 4

• helix-turn-helix
• zinc finger motif
• leucine zipper
• helix-loop-helix

Question 5

What is the structure of a helix-turn-helix binding motif?

L26 S22

Answer 5

• two connected α-helicies
• longer helix binds DNA
• binds DNA in dimers

Question 6

What is the structure of a zinc finger domain?

L26 S23-24

Answer 6

• binding domain contains a Zn atom

- found in tandem clusters on a single protein binding DNA multiple times

Question 7

What is the structure of a leucine zipper motif?

L26 S25-26

Answer 7

• two α-helix binding domain which grabs DNA

- dimerization occurs through interaction via leucines every seventh amino acid

Question 8

What is the structure of a helix-loop-helix binding motif?

L26 S27

Answer 8

-short α-helix chain connected to larger α-helix by a loop

Question 9

What is a gel mobility shift assay/electrophoretic mobility shift assay (EMSA) and how are they used to identify transcription factors?

L26 S38-39

Answer 9

• uses radioactive regulatory DNA fragment mixed with protein extract
• proteins bind DNA and then run on a gel
• DNA bound by proteins will separate based on size of protein
• proteins can then be isolated

Question 10

What is affinity chromatography and how is it used to identify transcription factors?

L26 S40

Answer 10

• column substrate is coated with matrix containing DNA sequences
• DNA binding proteins will be isolated from non-DNA binding proteins due to affinity for column substrate

-specific DNA sequences can be used to isolate specific transcription factors

Question 11

What is chromatin immuno-precipitation (CHIP) and how is it used to identify DNA binding sequences?

L26 S41

Answer 11

• formaldehyde is used to fix DNA associated proteins to DNA
• cell is lysed and DNA is fragmented
• DNA fragments bound to proteins are isolated and amplified using PCR

Question 12

What 4 methods are used to modify chromatin structure for increasing accessibility to DNA?

L26 S47

Answer 12

Nucleosome remodeling:
-sliding of nucleosomes to allow access

Nucleosome removal

Histone replacement:
-replacement with histone variants that allow greater access to DNA

Histone modification:
-destabilizes histone structure making histone easier to remove

Question 13

Which ways can gene repressors inhibit transcription? (6)

L26 S48-53

Answer 13

Competitive DNA binding

Masking activating domain

Interaction with transcription factors

Chromatin remodeling:
-tightens chromatin structure

Histone deacetylation:
-harder to remove deacetylated histones

Histone methylation:
-methylated histones bind proteins protecting tight histone structure

Question 14

How are gene regulatory proteins controlled? (7)

L26 S55

Answer 14

• synthesis
• ligand binding (activates)
• covalent modification (activates)
• addition of subunit
• unmasking
• nuclear entry
• proteolysis (cleavage from membrane protein)