MBB 331 - Midterm 3

what is chromatin?

eukaryotic DNA in complex with masses of protein (histones)

what are histones?

proteins that help chromatin fold to be packaged into the nucleus

what is DNA that is wound around histones called?

nucleosomes

how much DNA does a eukaryotic chromosome contain?

~2m long in humans

what are the three levels of DNA condensation?

- nucleosome: basic unit

- chromatin: beads on a string, conformation most of the time

- chromosome: most condensed

when would you find DNA compact into chromosomes?

during meiosis or mitosis (dividing cells)

how many bp of DNA fit into a nucleosome?

200 bp, including core and linker DNA

t or f:

the bacterial chromosome also contains nucleosomes

false

it is bound by proteins, but does not form nucleosomes

what are considered the building blocks of chromosomes?

nucleosomes

how would you determine nucleosome repeat length experimentally?

- micrococcal nuclease (not affected by cell age or culturing state)

- DNA bound by histones cannot be cut

- incomplete digestion results in fragments differing in 200 bp

- length of nucleosome repeat must be 200 bp

what are the 5 histones?

- H1 (linker)

- H2A, H2B, H3, H4 (core)

what histone varies among organisms?

H1

what do the core histones share?

common structural fold, the regions of the histone-fold motif form alpha helices (cylinders)

what composition of amino acids do histones have?

lysine and arginine because they are abundantly positively charged

how is a nucleosome assembled?

- initiated by H3(2)-H4(2) tetramer

- binds to dsDNA

- tetramer recruits two copies of H2A-H2B dimer to complete

how are the tails of histones removed?

treated with protease which specifically remove the amino terminal tails (because they are accessible) that leave the core intact

how do we know that DNA wraps around the histone core?

crystallography

what kind of wrap does the DNA do around the histone core?

left-handed solenoidal supercoiling around the histone-fold motif

what positions do the histone tails emerge from the core?

- H3 and H2B emerge between the two DNA helices

- H4 and H2A emerge from either below or above the double helix

what is the histone fold motif?

where the DNA wraps around

what are the histone tails?

N terminal and C terminal

what part of the DNA do the core histones make contact with?

the minor groove

t or f:

histone tails contribute to DNA binding

false

they don't contribute to DNA binding but mediate inter-nucleosome connections that affect chromatin structures (compaction level)

what does the structural change of chromatin rely on?

the modification of histone tails which affect DNA replication, repair, and transcription (epigenetics)

what does the addition of H1 cause?

more compact nucleosomal DNA, the core histones and H1 protect a larger amount of DNA meaning there is greater compaction

how many binding sites does H1 have?

two DNA binding sites, one that binds to linker DNA and the other that binds to the DNA wrapping around the core histones

what kind of pattern does H1 create in DNA?

zigzag pattern by bringing the entry and exit very close together

what is 30 nm fiber/filament?

higher order of organization of nucleosomes thought to exist in living cells

what are the two models of the 30 nm fiber?

- solenoid model: one-start helix

- zigzag model: two-start helix (not double stranded)

what form are sites of active DNA in (transcription and replication, etc)?

10 nm fibre ("naked" DNA)

what could you treat 30 nm fiber with to expand them?

low salt buffer to reveal the loops

what is the majority of DNA packaged into?

large loops of 30 nm fiber that is tethered to the chromosome scaffold at their base

why is scaffolding needed in DNA?

because there is so much DNA in eukaryotic organisms that its packaging needs organization, provided by scaffolds

how is bacterial DNA organized?

since it lacks nucleosomes, its compacted into looped domains attached to one or more points on the inner surface of the plasma membrane mediated by SMC proteins that organize the circular chromosome into a scaffold like structure

what controls accessibility to DNA?

nucleosomes control the accessibility of DNA to transcriptional machinery (RNAP and regulatory proteins)

what are the "open" and "closed" chromatin states associated with?

- "open" active gene transcription

- "closed" transcription repression

what are the characteristics of nucleosome free zones?

control regions of active genes and are DNase hypersensitive

what is DNase hypersensitivity?

corresponds to the presence of gene-specific factors (transcription factors) that exclude nucleosomes from active genes, but not from inactive genes

what occurs if chromatin is treated with DNase I?

digested DNA is isolated to see the distance from the known restriction site to determine the hypersensitive site

- inactive gene: no DNase hypersensitivity

- active gene: DNase hypersensitive regions are digested

how can repression caused by H1 be counteracted?

by transcription factors such as Sp1 and GAL4 which are anti-repressors of histones, they remove nucleosomes that obscure a promoter or prevent the binding of nucleosomes to promoters in the first place, and activators of transcription

what is nucleosome positioning?

activators force the nucleosome to take up positions around, but not within the promoter

t or f:

in many cases it is critical for nucleosomes to be moved or loosened from DNA

true

this is to allow other proteins to have access to the DNA and allow expression of the genome

what are the classes of enzymes that regulate nucleosome arrangement?

- chromatin remodelling complexes: ejecting (activate) or reposition/replacement (depends, may activate)

- histone modifying enzymes: covalent modification of the amino (N) terminal tails of histones

what is the mechanism of chromatin remodelling complexes?

the chromatin remodelling complexes pull the DNA out of the nucleosome into a loop to expose the promoter

what modifications to histone tails (N terminal) are made?

acetylation and phosphorylation

what does acetylation and phosphorylation do to histone tails?

neutralizes the positive charge to reduce affinity of tails for the negatively charged backbone of DNA

what does the modification of histone tails cause?

- reduction of overall positive charge

- affects the ability of nucleosome arrays to form more repressive higher order chromatin structure (more condensed)

what enzymes are used in histone tail modifications?

- HAT: histone acetyltransferase

- HDAc: histone deacetylase

- HMT: histone methyltransferase

- HDM: histone demethylase

what does histone deacetylase do?

"tightens" DNA binding to nucleosomes causing more gene repression/silencing

how are histone deacetylases activated?

transcription repressors (nuclear receptors without their ligands) interact with corepressors which interact with histone deacetylases that remove acetyl groups from the basic tails of core histones to stabilize the nucleosome and reduce transcription

t or f:

the presence of a ligand (such as a hormone, like thyroid) causes transcription repression

false

the presence of a ligand will bidn the nuclear receptor meaning that HAT (histone acetyltransferases), a coactivator, will loosen DNA binding to histone cores and make it more accessible for gene expression

what are the significant targets of histone acetyltransferase and deacetylase?

the core histones, not H1

what are the effects of methylation of histone tails?

either activation or repression depending on what proteins are recruited by it (either a bromodomain or a chromodomain)

what are bromodomains?

allow proteins to bind to acetylated histones (activation of transcription)

what are chromodomains?

allow proteins to bind to methylated histones, commonly found in heterochromatin (repressed transcription)

what occurs if a chromatin remodelling complex contains a bromodomain in a subunit and HAT activity?

the complex binds to an acetylated nucleosome and acetylates the histones in the neighbouring nucleosome to propagate a pattern of acetylation, leading to higher levels of gene expression

what interaction causes the crosslinking/tight packing of nucleosomes?

the histone tail (N terminal - basic) of H4 interacts with the acidic pocket in the H2A-H2B dimer in the adjacent nucleosome, repressing transcription

how does the acetylation of H4 tails activates transcription?

neutralizes the positive charge of the N-terminal tail, prevents nucleosome crosslinking to stop the repression fo transcription

why is acetylation of the core histones not enough to permit transcription factors access to DNA?

it is essential, but not sufficient because the nucleosomes are still intact so something needs to remodel the nucleosome cores

what is conserved between all three classes of chromatin remodelling complexes?

ATPase domain, they use ATP to disrupt the contact between nucleosomes and ANDA to alter chromatin structure

how would histone modifications NOT be inherited?

if histone octamers were completely displaced during replication, the epigenetic information encoded in histone modifications would be lost

what are histone chaperones?

assist in preserving epigenetic information encoded in histone modification

how are histone modification inherited?

half the octamer (H2A-H2B) are displaced while the other half is retained, the displaced half stays nearby for new assembly (not degraded)

t or f:

the spread of epigenetics states remain after replication

true

if it is closed or open, half of the nucleosomes will have the original modifications to be passed onto the new ones

what is epigenetic inheritance?

inheritance not encoded in DNA sequence

t or f:

regulation of chromosome structure is not essential for all types of genetic processes, but is essential for transcription

false

it is essential; for all types of genetic processes (transcription, replication, repair, and recombination)

what pathway does acetylation lead to activation of transcription?

histone code

what are the types of histone modification?

- acetylation

- methylation

- phosphorylation

- ubiquitination

what effects can chromatin remodelling complexes do to nucleosomes?

- change position of nucleosomes

- eject histone octamers

- replace canonical histones with histone variants

what is the model for deacetylation of histone tails?

removing acetyl groups leads to RNAP dissociation and chromatin condensation

- repressors displace activators

- corepressor inhibits RNAP

- HDAc associates with corepressor, RNAP dissociates

- deacetylation leads to chromatin condensation and transcription repression

t or f:

nucleosomes will occupy any position throughout the genome

false

they have favoured positions usually regions that are largely conserved and depleted at control regions (enhancers, promoters, terminator regions)

how can nucleosome positioning be determined?

- digest DNA with nuclease

- immunoprecipitate nucleosome-DNA with histone antibody

- reverse nucleosome-DNA crosslinks

- identify by DNA sequencing

- label DNA with fluorescent group and anneal/probe microarray

what is the difference between where transcription and translation occurs in bacteria and eukaryotes?

- in bacterium, transcription and translation are couples

- in eukaryotes, transcription occurs in the nucleus and translation occurs in ribosomes in the cytoplasm

what are bacterial genes controlled by?

repressor and RNAP

why are transcription start sites (TSSs) in eukaryotes unique compared to bacteria?

they have diverse distances from transcription factor binding sites across genes (compared to conserved -10, -35 in bacteria)

how can differentiated cells be converted to another cell type?

by manipulating the expression of transcription factors

what RNA products comes from Pol I?

18S, 28S, 5.8S rRNAs

what RNA products comes from Pol II?

mRNA, microRNAs, noncoding RNAs

what RNA products comes from Pol III?

tRNA, 5S rRNA, 7SL RNA

what is the most relevant RNAP in eukaryotes?

RNAP II (Pol II) made of 12 subunits

where is the DNA template accepted into RNAP II?

the deep cleft where 2 Mg2+ molecules act within the catalytic core (RPB 1)

t or f: the bacterial RNAP and eukaryotic RNAP II are not similar in structure but are in sequence homology

false

both structure and sequence homology are similar

how were the subunits of RNAP II isolated?

using epitope tagging one subunit of the whole complex (coimmunoprecipitation)

- radioactively labelled proteins (35S for proteins, 32P for phosphorylated subunits (activated))

- antibody against epitope added

- precipitate antibody bound to complex (coimmunoprecipitation)

- resolve bound proteins on SDS-page gel

what subunits have homology to the bacterial RNAP subunits? why is this important?

RPB1, RPB2, RPB3 (core subunits) share homology to beta, beta', and alpha

all are required for activity and have fundamental roles in transcription

t or f:

all eukaryotic RNAPs are regulated in the same way

false

each eukaryotic RNAP binds to specific promoter sequences that control the expression of their respective RNAs

what is similar between all three RNAPs in eukaryotes?

they use a factor called TBP (TATA-binding protein) which plays a role in transcription initiation

t or f:

all genes have TATA boxes, without these TBP cannot be recruited

false

not all genes have TATA boxes, the genes without recruit TBP through TAFs (TBP-associated factors) that recognize promoter sequences

what composes the RNAP I preinitiation complex?

- RNAP I

- one SL1 (selectivity factor 1, a complex made of one TBP and 3 TAFs)

- two UBFs (upstream binding factors, for high transcription)

where are the promoters of RNAP III located?

they are found within genes

t or f:

RNAP III have TATA boxes

false

what order do they tRNA promoters and 5S rRNA promoters bind transcription factors (RNAP III)?

tRNA: TFIIIC first, recruits TFIIIB (contains TBP)

5S rRNA: TFIIIA, recruits TFIIIC, recruits TFIIIB (contains TBP)

what composes the RNAP II preinitiation complex?

- general transcription factors (GTFs)

- RNAP II

- promoter

what are enhancers?

regulatory sequences (DNA) thousands of bp (upstream or downstream) from the core promoter

what is an Inr?

initiator sequence (weak consensus)

where is the TATA box found in eukaryotes? what is unique about this?

located 26-31 bp upstream from the start site (-26 to -31), it is the only element with a fixed position found in all eukaryotes

what order do the transcription factors bind in RNAP IIA?

1. TBP or TFIID (in vivo also needs TFIIA and mediator complex)

2. TFIIB

3. TFIIF (on the back of RNAP IIA)

4. TFIIE

5. TFIIH (helicase and kinase)

what role does TFIIA have in RNAP IIA?

unmasks TBP to increase binding efficiency (stabilize TBP-TATA interaction) and protects upstream

how can promoters be studied?

using linker scanning mutagenesis

- cut, digest, and insert linker (shifts regulatory sequences)

- if the TATA box is disrupted, the gene is not transcribed

- when run on a gel, there will be a gap in the bands compared to the control (wild-type) meaning no transcription and shows position

what is the difference between prokaryotic transcription and eukaryotic transcription?

1. RNAP II requires GTFs (general transcription factors) for basal transcription

2. DNA binding transcription activators can control promoters (DNA binding proteins that act upstream of core promoters)

3. coactivators bind proteins for communication between RNAP, GTFs, and activators (can be cell/tissue/time specific or response to stimuli)

t or f:

there are defined terminators of transcription in eukaryotic transcription

false

there are no defined terminators