Lecture 8 Flashcards
Issues with cartoon version of central dogma
Nucleus not filled with DNA, DNA is not naked; its not all being transcribed, not does it contain all coding sequences.
mRNA and protein lifetimes
mRNA life times
E. coli: 5 minutes
yeast: 20 minutes
human cells: 10 hours
protein life times
yeast: 40 minutes
human cells: 2 days
Genome in eukaryotes
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.
Cell type
Determined by both spatial and temporal organisation; genome plays important but not exclusive role.
RNA polymerase II
Transcribes DNA into RNA
What controls transcription
Gene control regions
Promoters
Integrate multiple inputs - wealkly activating protein assembly, strongly inhibiting protein, strongly activating assembly, ===> probability of initiating transcription.
Master TFs
Specify cell types. Regulate complex gene expression programs.
Nucleosome
Non-selective. DNA is wrapped around them.
10e9 base pairs per genome. 1nm per base pair. == 1m per genome»_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.
Core histones
tightly associate w/DNA. because of + charge. So e- static interactions.
How do nucleosomes pack together
In higher order structure = compact chromatin fibre.
“30nm fibre”
Compact chromatin saves space…how does it affect transcription?
Nucleosomes prevent transcription factor binding.
Competing constraints on the three-dimensional organization of the
genome:
the genome must be compact enough to fit in the nucleus, but open enough for TFs to bind to the DNA.
So how do transcription factors access the DNA?
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.
Transcription factors direct local alterations of
chromatin structure
Heterochromatin
Heterochromatin is highly condensed and
restricts gene expression
% of chromatin open and active
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.
Covalent modifications of histone tails
Many diff sites. 2 classes of modifiers - writers (add functional groups) and erasers (remove functional groups).
histone code
determines nucleosome
stability and higher-order structure
Writersadd functional groups
Erasersremove functional groups
Readersbind specific histone
modifications
Reader” complexes
translate the histone code
Lateral propagation of chromatin condensation
Lateral propagation of chromatin condensation
Propagation of heterochromatin across generations
Epigenetic control of gene expression. Same histones will reassociated with the same DNA, but only 1/2 will be post-translationally modified.
Heterochromatin localizes
To the nuclear membrane
Gene-rich regions
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.
Chromosomes
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.
Interphase chromosomes fold into topologically associated domains (TADs).
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.
TAD formation
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.
Cohesin
an SMC protein complex
CTCF defines TAD boundaries: how do we know?
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.
Evidence that CTCF defines TAD boundaries
