mbii - l21 - transcription 5 - nucleosomes and chromatin
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8/8/2019 MBII - L21 - Transcription 5 - Nucleosomes and Chromatin
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Molecular Biology II Nucleosomes and Chromatin
Lecture 21[Page 1]
Eukaryotic promoters are more complex than those found in prokaryotes
Consist of:
The basal initiation complex, at the transcription start site (+1)
Proximal gene-specific transcription factors (~1kb upstream)
Distal gene-specific transcription factors (~1-100kb upstream)
DNA between these regions will be wound up as chromatin for organisation and compaction. This DNA will be made
inaccessible.
Nucleosome Fibre:
Nucleosome = Histone + DNA complex
The nucleosomes are regularly spaced and resemble µbeads
on a string¶.
Micrococcal nuclease will cleave (linker) DNA between each nucleosome, but will not cleave DNA that is bound to the
histones. Partial digestion will result in differing fragments containing various numbers of histones. This will produce
distinct fragments containing 1 or more repeated units that can be visualized by electrophoresis. (mononucleosome,
dinucleosome, tri-, tetra- etc.)
Nucleosome particles consist of 4 different polypeptides (core histones)
H4, H2A, H2B, H3 (In increasing weight order)
Histone Structure:
All 4 histones contain a central µhistone fold¶ (A)/(B)
The histone fold is a dimerization motif (C)
A very distinct fold (3D structure) found almost exclusively in histones
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@ Histones evolved early (2 billion years ago)
Have been largely unchanged since
Most of these species are hyperther mophiles (80r+) ± histones may have originally evolved to protect 8 NA in
extreme conditions
There is a very high degree of structure conservation between Archaeal and Eukaryotic histones
High resolution X 9 ray
crystallographic structurep
8 NA (yellow) loops around
nucleosome (blue) 1.7 times Nucleosome 3 8 Structure
146bp of 8 NA are bound
8 isk-like structure
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Molecular Biology II Nucleosomes and Chromatin
Lecture 21[Page 3]
Nucleosome-@ NA Interactions:
H-bonds & Ionic interactions
Non-polar contacts
Intercalation of Arg residues to contact phosphates across the groove (utilises AT bps)
Amide / phosphate bond interactions
Histone helix dipoles to spatially fix single phosphates
The strength of the interactions between the nucleosomal surface and A NA is required to bend the 146bp long A NA
into a circle (1.7x). This is nor mally highly energetically unfavourable due to the rigidity of ds A NA. Requires tight
binding, a large amount of non-covalent forces are required.
This will distort the A NA, and prevent nuclease cleavage.
The surface of the histone octamer is covered with many positively charged amino acid residues. They will interact
strongly with the negatively charged phosphate groups in A NA.
Linker histones (such as H1 / H5) stabilise the interaction between nucleosomes in compacted chromatin. They
organise the entry and exit points of A NA on the nucleosome. The presence of H1 extends the nuclease protection
from 146bp (core nucleosome) to 168-200bp.
Partially species dependent
Bind to linker A NA
Chromatin fibres are even more condensed than
nucleosomes alone. H1 linkers come together to for m the
30nm chromatin fibres.
This results in a complex solenoid (spiral-like) structure
p
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Molecular Biology II Nucleosomes and Chromatin
Lecture 21[Page 4]
B NA in this state is transcribable, the nucleosomes do not need to be removed, just unwound from the solenoid.
When tightly packed into the solenoid (30nm):
DNA storage for m
No gene transcription
High levels of histone H1
Many positively charged N-termini that bind C NA on neighbouring nu cleosomes
When unpa cked (11nm beads ):
More accessible for m
Reduced levels of histone H1
Gene transcription is possible
Many highly a cetylated core histones (espe cially H3 & H4)
Histone N-ter mini :
In addition to the conser ved histone f old , histones contain additional se quen ces , mostly at the
N-ter mini , that play an i mportant role in controlling the gene -reg ulatory roles o f n ucleoso mes .
These additional se quen ces are very f le xi ble and do not sho w up in X-ray crystal str uct ures .
There are f ar f e wer se quen ces at the C-ter mini
The N-ter mini can protr ude f ro m the core i f fully e xtended . They are f le xi ble .
Acetylation o f Conser ved Lysines :
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Molecular Biology II Nucleosomes and Chromatin
Lecture 21[Page 5]
The N-termini of H3/4 and their acetylation patterns are absolutely conserved
These K residues can be acetylated or methylated
Acetylation will remove the positive charge from lysine residues, which will stop them interacting with the phosphates
in the DNA backbone.
The enzyme HAT (Histone Acetyl-Transferase) adds the acetyl group, provided by acetyl-CoA to form -N-Acetyl-
Lysine
Histone deacetylase carries out the reverse reaction
In the deacetylated state, chromatin will be closed and tightly wound. The µtails¶ will be wrapped around the
nucleosomes.
Acetylation will result in the formation of open chromatin. The tails are fully extended. The DNA is more relaxed and
histones can be dislodged to allow transcription
Opening of Chromatin by Gene-Specific TFs:
Certain gene-specific TFs are still able to recognise their target sequence when DNA is chromatin packaged.
Upon binding, these TFs can recruit other enzymes such as HATs, to create a transcriptionally active chromatin
environment.
Alternatively HD ACs (Histone DeACetylases) to create a repressed chromatin environment.
GCN4 is a gene-specific activator. It can recruit HATs (such as GCN5) locally to create a loosened region that is
transcription capable. There will be poorer acetylation further away.
Ume6 is a gene-specific repressor which recruits HD ACs such as the Sin3 complex. Creates a hypoacetylated
chromatin environment which will be inaccessible to the transcription machinery.
Chromatin is essential for gene regulation, as the only other way to stop polymerase is via a µroadbloack¶.
Methylation:
An important post-translational control mechanism that can distinguish between transcriptionally active and passive
forms of chromatin.
Repetitive DNA near centromeres is densely packed in the form of heterochromatin.
Nucleosomes are methylated and contain special proteins including linker histone H1 to maintain high packing density.
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Molecular Biology II Nucleosomes and Chromatin
Lecture 21[Page 6]
Transcribed parts of the genome are present in the form of euchromatin.
Nucleosomes are acetylated, low level of H1 linkers. Loosely packaged.
Chromatin structures are not the ultimate control level of gene expression
The nucleus has a very sophisticated architecture that influences genome expression on a more global level
Transcription& splicing occurs within distinct regions
Chromosomes are found within distinct domains within the nucleus
Import / export through nuclear pores is tightly controlled
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