Lec 19 - Regulation of Gene Expression & Epigenetics

Question 1

chromatin & transcription

histone role in reg?

Answer 1

• histones can be moved around & placed at specific locations
• positioning of histones & nucleosome-free regions important for reg
• nucleosomes can interfere w/binding of polymerases and TFs to the DNA (making less accessible)

DNA wrapped around histones to facilitate neg supercoiling; 8 hostones make 1 nucleosome; writhe = -1.67

Question 2

nucleosome free promoters

why is this? differences in test tube?

Answer 2

• promoter DNA is nucleosome-free in cells
• allows access to general TFs to bind to nucleosome free region & assemble pre-initiation complex
• DNA & histones in test tube don’t do this, meaning moving histones is an active process (enzymes in cell actively remodel chromatin)

Question 3

chromatin remodeling

what is this? what complex involved?

Answer 3

• act of protein complexes rearranging number & position of nucleosomes
• use energy of ATP hydrolysis
• SWI/SNF is an important chromatin remodeling complex

Question 4

histone modification & SWI/SNF complex

Answer 4

histone modification
- histones have flexible tails that can be modified post-translationally; lysine can be acetylated (reversible)
- specific lysines within the histone tail are modified (eg H3K9, histone 3 at lysine residue 9)
- histone acetyltransferase (HAT) adds acetyl to Lys
- histone deacetylase (HDAC) removes acetyl from Lys

SWI/SNF complex
- proteins can bind to nucleosomes w/modified histone tails
- SWI/SNF complex binds specifically to H3K9-Ac & H3K14-Ac and helps space the nucleosomes
- lysine acetylation is associated with chromatin that is accessible for transcription; euk repressors can recruit HDACs to close chromatin

Question 5

heterochromatin vs euchromatin

Answer 5

• hetero: dense, no transcription (silent)
• euch: less dense, transcription (active)

Question 6

in heterochromatin:

lysine methylation

what happens? what 2 enzymes involved? how is this process contagious?

methylation vs acetylation

Answer 6

• specific lysine side-chains in histone tails are methylated in heterochromatin (dec transcription)
• histone methyltransferase (HMT) add methyls & histone demethylase (HDM) removes thm
• 1, 2, or 3 methyls per lysine; either methylation or acetylation could occur (mutually exclusive)

signal spreading
- heterochromatin has lysine methylation on histone 3, lysine 9; this position important bc acetylation at same position is signal for* transcriptional activation* (opp)
- HP1 (heterochromatin protein 1) binds H3K9 methyl & recruits SUV39H1, which further methylates histones (chain rxn, heterochromatin can spread)

• methyl comes from S-adenosylmethionine

Question 7

RNA-induced silencing

Question 8

siRNA biogenesis

Question 9

miRNA biogenesis

Question 10

euk mRNA decay

Question 11

reg. RNA Binding proteins

Question 12

proteasome

Question 13

ubiquitin

Question 14

proteosome lid

Question 15

recombinant DNA

Question 16

molecular cloning

Question 17

plasmids

Question 18

restriction endonucleases

Question 19

compatable ends; polylinkers

Question 20

cloning PCR products

Question 21

seamless cloning

Question 22

resection & annealing

Question 23

extension & ligation

Question 24

gibson assembly

Question 25

CRISPR

Question 26

site-specific recombination

Question 27

recombinases

Question 28

genetically engineered recombination