mbii - l21 - transcription 5 - nucleosomes and chromatin

8/8/2019 MBII - L21 - Transcription 5 - Nucleosomes and Chromatin

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Molecular Biology II Nucleosomes and Chromatin

Lecture 21[]

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




Lecture 21[]

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




Lecture 21[]

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 :




Lecture 21[]

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.




Lecture 21[]

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

mbii - l21 - transcription 5 - nucleosomes and chromatin

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