Lecture 8

Question 1

Issues with cartoon version of central dogma

Answer 1

Nucleus not filled with DNA, DNA is not naked; its not all being transcribed, not does it contain all coding sequences.

Question 2

mRNA and protein lifetimes

Answer 2

mRNA life times
E. coli: 5 minutes
yeast: 20 minutes
human cells: 10 hours

protein life times
yeast: 40 minutes
human cells: 2 days

Question 3

Genome in eukaryotes

Answer 3

Most of it is noncoding.

About 20,000 genes; in humans, alt. splicing means we have approx 70,000 genes, and with post-translational modifications we have about 1M proteins.

Question 4

Cell type

Answer 4

Determined by both spatial and temporal organisation; genome plays important but not exclusive role.

Question 5

RNA polymerase II

Answer 5

Transcribes DNA into RNA

Question 6

What controls transcription

Answer 6

Gene control regions

Question 7

Promoters

Answer 7

Integrate multiple inputs - wealkly activating protein assembly, strongly inhibiting protein, strongly activating assembly, ===> probability of initiating transcription.

Question 8

Master TFs

Answer 8

Specify cell types. Regulate complex gene expression programs.

Question 9

Nucleosome

Answer 9

Non-selective. DNA is wrapped around them.
10e9 base pairs per genome. 1nm per base pair. == 1m per genome&raquo_space; 10 um nuclear diameter.

Nucleus has packing problem. so, genome is organised in chromatin, which is protein + DNA. about 1/3 NA, 1/3 protein.

DNA winds around histone octamers.

Question 10

Core histones

Answer 10

tightly associate w/DNA. because of + charge. So e- static interactions.

Question 11

How do nucleosomes pack together

Answer 11

In higher order structure = compact chromatin fibre.
“30nm fibre”

Question 12

Compact chromatin saves space…how does it affect transcription?

Answer 12

Nucleosomes prevent transcription factor binding.

Question 13

Competing constraints on the three-dimensional organization of the
genome:

Answer 13

the genome must be compact enough to fit in the nucleus, but open enough for TFs to bind to the DNA.

Question 14

So how do transcription factors access the DNA?

Answer 14

Nucleosomes are dynamic.

Passive mechanism: They “breathe”. Histone and DNA bind and separate. The open form occurs about 1/20th of the time. Breathing is random passive movement. Cooperative effect - one TF regulator can bind and stabilise the open form, facilitating the binding of another TF regulator.

Active mechanism: ATP-dependent chromatin remodelling complexes. Nuclear turnover.

Question 15

Transcription factors direct local alterations of
chromatin structure

Answer 15

Question 16

Heterochromatin

Answer 16

Heterochromatin is highly condensed and
restricts gene expression

Question 17

% of chromatin open and active

Answer 17

Only 20%. Not all euchromatin is transcriptionally active.
Heterochromatin is inactive, but can still be modified to become transcriptionally active.
Constitutive heterochromatin is permanently inactive - centromeres, telomeres, highly compact.
Facultative heterochromatin is regulated - varies across cell type.

Question 18

Covalent modifications of histone tails

Answer 18

Many diff sites. 2 classes of modifiers - writers (add functional groups) and erasers (remove functional groups).

Question 19

histone code

Answer 19

determines nucleosome
stability and higher-order structure

Writersadd functional groups
Erasersremove functional groups
Readersbind specific histone
modifications

Question 20

Reader” complexes

Answer 20

translate the histone code

Question 21

Lateral propagation of chromatin condensation

Answer 21

Question 22

Lateral propagation of chromatin condensation

Answer 22

Question 23

Propagation of heterochromatin across generations

Answer 23

Epigenetic control of gene expression. Same histones will reassociated with the same DNA, but only 1/2 will be post-translationally modified.

Question 24

Heterochromatin localizes

Answer 24

To the nuclear membrane

Question 25

Gene-rich regions

Answer 25

loop out from chromosome territories
Genes occupy larger volume when they’re being transcribed. - highly decondensed, form larger loops, occupy larger volume of the nucleus.

Question 26

Chromosomes

Answer 26

Occupy distinct neighbourhoods - diversity of cell types? Sequences close together within the nucleus even if not on approximate portions of DNA.

Experiment - cross linking protein to DNA, cut with restriction nuclease, mark ends with biotin, ligate DNA, remove terminal biotin and purify biotin containing DNA, adding linkers to DNA ends, test for joined segments by PCR and sequencing.

Question 27

Interphase chromosomes fold into topologically associated domains (TADs).

Answer 27

Signal intensity should fall off the diagonal line if DNA randomly folded. If DNA didn’t fold up at all, would only see diagonal line. Many sequences are ligated to sequences far away. Chromosomes fold to form neighbourhoods.

Question 28

TAD formation

Answer 28

An insulator protein complex (CTCF dimer complex - binds specific sites, defines boundary) and a molecular
motor (cohesin - walls a;pmg DMA and causes to tp ;pp[ pit) contribute.

Question 29

Cohesin

Answer 29

an SMC protein complex

Question 30

CTCF defines TAD boundaries: how do we know?

Answer 30

Correlation - TAD boundaries should correlate with CTCF DNA binding sites.

Necessity - Removal/inhibition of CTCF should abolish TADs.

Sufficiency - Introduction of ectopic CTCF binding sites should redefine TADs.

Question 31

Evidence that CTCF defines TAD boundaries

Answer 31