Histones and Packaging Flashcards
Why does DNA need to be packaged?
So biological processes can be carried out
How many bases does one complete double turn of double-helical DNA contain?
10
Why does DNA need to be unwound?
To allow access to machinery for replication and transcription
How many bases does the human haploid genome contain?
3 x 109 bases
Approximately how much DNA sin each diploid cell?
2m
What is the role of chromatin?
To package DNA
What is chromatin made up of?
Nucleosomes (which are made up of histones)
What factors do histones affect?
Isolation
Characterisation
Biochemical properties
What is the composition of chromatin?
DNA
Proteins (mainly histones)
RNA (not coding for proteins)
What is the most common nuclear protein?
Histones (very abundant)
What is the role of histones?
Packaging
What is the ratio between histones and DNA
1:1
What is the non-histone chromatin protein used for?
Scaffolding
What is the role of RNA?
Used when the nucleosome is acting as a signalling molecule to either allow or prevent transcription
What are the core four histones?
H4 H3 H2A H2B
What is the linker histone?
H1
Which is the least conserved histone?
H1
What are the most highly conserved histones?
H4 H3
What are the properties of histones and how do they help carry out their role in packaging?
-Very small
-V highly positively charged
-highly conserved
Ideal for packaging as DNA is v negatively charged
How many amino acids difference is there in H2A and H2B
2
What is the first level of packaging?
10nm fibre
What’s the second level of packaging?
30nm solenoid
What does the nucleosome consist of?
2 x H2A 2 x H2B 2 X H3 1 H1 200bp DNA
What determines the packaging of DNA?
How the histones interact
What is the canonical nucleosome?
The normal contribution of histones
How many base pairs of DNA fit in the nucleosome?
146 base pairs of DNA left handed super helix
How many base pairs of DNA wraps around the nucleosome?
146
What happens to the other base pairs that are not wrapped around the nucleosome?
The extra DNA either side of the nucleosome is used to link one nucleosome to another nucleosome (only time DNA is in a naked state)
What is the total protein content of the nucleosome?
103Kda
How does the heterodimerisation of histones H3 and H4 occur?
Histone handshake motif
2 molecules of each histone bind as a tetramer
Forms a horseshoe shape
How do H2A and H2B associate with the complex?
They form dimers above and below the tetramer
What could a change any of the four histones cause?
It would change the interaction of dimers which could change the core octameric structure which could make it less stable
How does DNA wrap about the octameric core?
It wraps around in a left handed super coil (even though DNA is a right handed helix)
DNA does not run smoothly around the nucleosome (giving rise to pockets)
What is the role of the pockets?
Allow accessibility of transcription factors and chromatin remodelling factors to remodel how DNA is taking its path around the nucleosome
What happens if the canonical nucleosome is altered?
The path taken by the DNA will change which is vital during transcription and transcription because DNA needs to be used as a template so must be able to unravel from the nucleosome
How many variants does H2 have (and name them)?
At least four H2AZ macroH2A H2AX H2A.Bbd
When would histone variants be used?
if you want to activate or suppress gene transcription by changing the canonical nucleosome
What is the role of H2AZ?
Used to increase gene activity
What is the role of macroH2A?
Placed in nucleosome on inactive X chromosome (one X chromosome is turned off so will be enriched in macroH2A to keep it turned off)
What is the role of H2ABbd?
Placed on active X chromosome so keep it active
What is the role of H2AX?
Signals to repair broken DNA so it’s not transferred into the next generation
What percentage identity does H2A have with H2AZ?
60% (quite low)
How does the use of H2AZ instead of H@A increase gene transcription?
- Amino acids are different so the interaction stability is lowered between H2AZ and H2B
- Alters the interaction of the H2A/H2B dimer with the H3/H4 tetramer
- DNA wrapped around nucleosome not as strongly so transcription can easily occur
- H2AZ tail is also longer which affects interactions in central core
How many variants are there of H3?
5 (but very little difference between them)
What are the variants of H3?
H3.3 H3A H3.2 CenpA H3.lt
What is the role of CenpA?
It is enriched at the centromeres and telomeres where there are no genes so you can attach the chromosome or at the telomers to protect the genome
What are histone variants used for?
To alter the packaging of DNA
What does replacement of H3 with H3.3 cause?
marks actively transcribed loci by replication independent nucleosome assembly
What do variants of H3 and H2A differentiate between?
chromatin at centromeres, active genes and heterochromatin
When is there no gene expression?
Metaphase (most highly condensed state)
Describe the 10nm fibre
A series of nucleosomes linked by linker DNA
What is the packing ratio in the 10nm fibre?
6-7
What does the length of the linker DNA depend on?
cell type and species
Describe the structure of the 30nm solenoid?
- 10nm coiled
- histone H1 binds and stops the DNA from slipping
- 6 nucleosomes per turn
- packing ratio of 40
Describe the structure of the 300nm solenoid?
- Each loop contains between 60-100 kb of DNA tethered by non-histone scaffolding proteins
- protein scaffolding allows the DNA to condense further
- non-histone proteins keep space between loops and prevent crossing
- packing ratio of 680
Describe the structure of the 700nm fibre
- loops of chromatin coil again to form a coiled coil
- scaffold has loops of 30nm loops
- packing ratio of 10^4
What is the level 6 of packaging?
- metaphase chromsome
- whole of DNA completely inaccessible to transcription or replication machinery
What levels of packaging can transcription occur at?
Up to 30nm as long as transcription factors can associate
What are the two types of chromatin?
Heterochromatin and euchromatin
Where is heterochromatin present?
Telomeres and centromeres
Describe heterochromatin
Very highly compacted, very rarely contains genes within in it (usually a gene poor region)
In a interphase cell how would you distinguish between heterochromatin and euchromatin?
Heterochromatin- dense staining regions (highly compacted)
Euchromatin-light staining regions
What are the two types of heterochromatin?
Constitutive and facultative
What types of genes are contained within constitutive heterochromatin?
Poorly expressed genes
What is facultative heterochromatin often associated with?
morphogenesis or differentiation
Why don’t you want to put a gene into heterochromatin?
Because once its there you can’t transcribe it
Is constitutive heterochromatin the same or different in every cell?
same
Does facultative stay the same or change?
Can change
When is heterochromatin replicated?
Late in S phase
What type of chromatin configuration does euchromatin have?
Open configuration
When is euchromatin replicated?
Early in S phase (more accessible to replication machinery)
What types of gene does euchromatin contain?
Transcriptionally active and inactive genes
