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47 Cards in this Set
- Front
- Back
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Chromatin
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DNA + Proteins
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How does DNA fit into a nucleus?
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`10,000-100,000 fold reduction length
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what is the typical nucleus length? for humans?
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- Typical nucleus is 10 μm (1 x 10-5 m)
- Length of DNA in 1 human diploid genome is ~ 2.4 m (assuming 3.5x109 bp/haploid genome) |
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Histones
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- small, basic proteins (+)
* rich in arginine and lysine - highly conserved, abundant - must easily come on/off DNA - encoded by multiple genes - modify protein & regulate gene expression with positioning - can prevent transcription |
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what is an octamer? how many times does DNA wrap around it? how many linker DNA?
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- Two of each of the core histones
(H2A, H2B, H3, and H4) ~1.75 times ~ 80 bp turn = 146 bp wrapped around each octamer ~ 50 bp linker DNA |
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how many bp of DNA is there per nucleosome?
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~ 200bp
= ~70 nm linear |
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how many histones are there? what are they?
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5
• Core: H2A, H2B, H3, H4 • Linker: H1 |
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How many orders of packaging are there?
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3
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what are the beads/ "strings on a bead?
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- chromatin that has been treated with high salt to remove loosely associated proteins
- histone core + DNA |
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whats the 1st order packaging?
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- the nucleosome
" beads on a string" Histone core + DNA |
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Histone Tails
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- have the histone Code
- make it hard for nucleosomes to come close to each other -subject to various post translational modifications |
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histone code
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governs how DNA is packaged
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whats the 2nd order of packaging?
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- 30 nm fiber = -6-7 fold compaction
- solenoid - requires H1 |
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solenoid
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- a helix with 6 nucleosomes per turn
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what does histone H1 interact with and do?
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- interacts with linker DNA
- forces DNA to wrap around the core at the same position on each bead |
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Evidence for nucleosome structure?
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1. EM images "beads on a string"
2. Mild nuclease digestion of chromatin releases a nucleosome "ladder" of 200 bp 3. complete digestion of chromatin with DNase releases 146 bp band |
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How do you probe chromatin structure?
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- DNase to make linker DNA more accessible
- Partial digestion with DNase results in a ladder of ~ 200 bp bands |
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whats the 3rd order?
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- higher order chromatin folding
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higher order chromatin folding
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- 30 nm fiber is organized into loops
- 10- 100 kb per loop, anchored to non-histone protein scaffold |
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whats the impact of chromatin on gene expression?
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- DNA is not easily accessible
- chromatin must be unpacked for transcription and replication - controls gene expression |
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what do nucleosomes do?
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- prevent transcription
- regulate gene expression |
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what can nucleosomes prevent? evidence?
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- transcription
- in vitro reconsitution of chromatin a. naked DNA + core histones: 4-fold reduction b. +H1: 25 - 100- fold reduction |
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how does nucleosome positioning may regulate gene expression?
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- nucleosome covers promoter: no transcription even if TF is present
- some TF can remove nucleosomes or prevent their formation |
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what allows post translation histone modification?
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Histone tails
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four ways to modify histones
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important for structure/function & is reversible
- actylation - methylation - phophorylation - ubiquitination |
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Acetylation/ Lysine Acetylation/ Histone acetylation
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- relaxes chromatin
-Activate the gene expression - reduces + charge of histone tail - reduces affinity for DNA - loosens association of nucleosomes with a promoter |
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Modification of histones?
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- reversible reaction
- core histones can be acetylated on lysine residues |
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What enzymes modify histones?
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HAT - histone acetyltransferase = ON
HDAC - histone deacetylase = OFF |
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Methylation
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methylation = on
demethylation = off mono, di, tri on each lys |
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Methylation of H3 K4
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- associated with active genes
- demethylation = OFF/repressed |
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Methylation of H3 K 9
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- associated with silenced genes
- demethylation = ON/ gene activation |
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Influences Of Transcription Factors On Chromatin Structure
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- modify histone acetylation
- bind to DNA before histones bind DNA (after DNA rep) = pre - empting nucleosome formation - displaced chromatin after assembly -have HAT/HDAC activity |
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DNase Sensitivity
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- method to study chromatin structure
- if a gene is actively transcribed = less tightly packed in chromatin = more sensitive to DNase |
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DNase Sensitivity Steps
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1. Isolate intact nuclei
2. Partial digest with DNase 3. Purify naked DNA 4. Digest with BamHI/RE 5. Southern blot 6. Probe with gene |
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transcription factors vs histones
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1. pre-emptive model
a. TF + gene = gene active b. histone + DNA = nucleosomes = gene repressed 2. dynamic model a. TF knocks off repressor nucleosome and binds to promoter = active |
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chromatin remodeling
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- method to control gene expression
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Alternation of epigenic regulation
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- RNAi
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RNA interference gene silencing discovery
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- mainly after transcription
1. plants: silencing discovery cosuppression: plants 2. neurospora crassa 3. c. elegans |
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Silencing
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1. transcriptional level
- epigenetics: DNA methylation, histone modification 2. post transcription -RNAi down regulates= represses/degrades mRNA -small RNA: inhibits translation |
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Plant antiviral self defense
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RNA virus > host cell > replicates > dbRNA (trigger gene silencing) > RNAI > dicer> small RNA >RISC > viral genome/subgenomic RNA > viral RNA degradation
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db RNA
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intermediate trigger
1. plant 2. neurospora |
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dicer
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dbRNA > small RNA
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small RNA
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-can read DNA or RNA & sense/antisense strands
-20-30 nt - final noncoding trigger of gene silencing -derived from dsRNA -initiators of RNAi -2 different products |
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functions of small RNAs
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1. defense: antiviral/antibacterial
2. genome stability 3. development and cellular process 4. DNA elmination |
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differences btwn microRNAs and small interfering RNAs
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1. biogenesis pathway
2. precursors |
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microRNAs
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db RNA > ssRNA > folding into hairpin like > dicer (DCL) > RNA inducing silencing complex (RISC) > loads small RNA to look for targets in mRNA (cytoplasm) > anneals > repress translation or degrade mRNA changing mRNA level
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small interfering RNAs
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db RNA > RCR >DCL > RISC > induces gene silencing
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