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Y2 Genomes > Eukaryotic Genome > Flashcards

Eukaryotic Genome Flashcards

1
Q

What packages interphase DNA into chromatin?

A

Histones
Non-histone proteins
RNA

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2
Q

What is the challenge of compaction for DNA?

A

DNA must fit in the nucleus, but be accessible to transcription factors at molecular level

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3
Q

Which conditions is chromatin extracted under?

A

Low salt, so it has a more condensed form (30nm)
Without Mg2+

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4
Q

What are formed when chromatin is digested by nucleases?

A

Nucleosomes

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5
Q

Describe nucleosome structure

A

Octameric core
2 H2A/H2B heterodimers
2 H3/H4 heterodimers
Core histones have a central folded domain
147bp DNA makes 1.7 left-handed turns around the core

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6
Q

What is the histone variant H2AX used in?

A

DNA repair

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7
Q

What is the histone variant CENP-A used in?

A

Centromere DNA

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8
Q

What models do 30nm chromatin fibres fit?

A

Solenoid model: coil of nucleosomes
Zip-zag rope: Two rows of nucleosomes with DNA zip zagging between them

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9
Q

What is chromosome painting and how is it done?

A

Showing localisation of chromosomes. This is done by probing them with FISH

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10
Q

What sort of chromatin structure do actively transcribed genes have?

A

Looped out DNA. Associated with RNA polymerase and transcription factors

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11
Q

What are topologically associated domains (TADs)?

A

Subregions of chromosomal territories that are regulate gene expression by restricting regulatory elements to their genes
Separated by matrix associated domains

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12
Q

How are TAD boundaries established?

A

CTCF and cohesin which allows gene looping.
TADs are also flanked by LADs, which contain silenced DNA

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13
Q

Describe core histone tails

A

Flexible tails on histones which are post-translationally modified
Modifications can be mutually exclusive or be a prerequisite for another

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14
Q

Give an example of a mutually dependent post-translational modification

A

Ubiquitylation of H2B is required for methylation of H3K4

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15
Q

What types of post-translational modifications are there?

A

Acylation, methylation, phosphorylation and ubiquitylation

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16
Q

How is the positive charge on lysine ε-amino groups neutralised for histone modifications?

A

Histone acetyltransferases
This modification is reversed by histone deacetylase complexes (HDACs)

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17
Q

How can acetylation of lysine residues be blocked without affecting the positive charge?

A

Modification by histone methyl transferases (HMTs)

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18
Q

How are methyl groups removed from lysines?

A

Lysine-specific demethylases (LSDs)

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19
Q

How does acetylation make DNA more readily transcribed?

A

Acetylation takes away the positive charge from lysine, reducing histone interactions

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20
Q

What sort of chromatin is active?

A

Euchromatin

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21
Q

What sort of chromatin is inactive?

A

Heterochromatin

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22
Q

What are markers of actively transcribed DNA/ euchromatin?

A

H3K9Ac
H3K4me

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23
Q

What are markers of heterochromatin?

A

H3K9me
H3K27me

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24
Q

Outline the mechanism of the histone acetyltransferase complex, SAGA

A

1.SAGA is recruited to UAS elements by transcriptional activators (Gcn4 and Gal4)
2. Histones are acetylated , loosening nucleosome interactions and providing recognition sites for proteins with bromodomains
3. Negative charges are removed from phosphate backbones and DNA is transcribed

25
Q

Why are chromatin remodelling complexes needed?

A

DNA binding proteins cannot reach DNA that has associated with nucleosomes with ATP-dependent helicases

26
Q

How do chromatin remodelling complexes (CRM) work?

A

SWI/SNF have helicase activity which pushes DNA into nucleosomes, causing them so slide along
They have a bromodomain-containing subunit to bind to acetylation
These also function as co-activators/co-repressors

27
Q

How are histones deacetylated?

A

Sin3/Rpd3 histone deacetylase complex (HDAC) is a corepressor.
This is recruited to upstream regulatory sequences (URS) by interacting with the Ume6 repressor domain.

28
Q

How does heterochromatin protein 1 (HP1) link nucleosomes in heterochromatin?

A

HP1 links nucleosomes containing H3K9me. The chromodomain of HP1 binds methylated histones
Nucleosome interactions can be mediated with chromodomain shadows which interact with each other
Nucleosomes carrying H3M9me can be drawn together

29
Q

How does Suv 3-9 (histone methyltransferase) stimulate methylation on H3K9me3?

A

The Suv 3-9 chromodomain binds to H3K9me3 and stimulates SET activity (methylation catalysis) on the histone.
Adjacent methylated histones are linked by HP1

30
Q

How does heterochromatin form in yeast?

A

Sir2 (histone deacetylase)
Sir3 (nucleosome binding protein)

31
Q

How is spread of heterochromatin formation stopped in eukaryotes?

A

Eukaryotes: limited by chromatin boundary elements
Insulator sequences are clusters of binding sites for non-histone DNA binding proteins

32
Q

How is spread of heterochromatin formation stopped in yeast?

A

Yeast: Gene insulators stop heterochromatin forming in actively transcribed genes

33
Q

What can be used to assay nucleosome regions?

A

ChIP assays

34
Q

What can be used to assay protein binding to DNA?

A

ChIP assays

35
Q

Outline nuclease sensitivity assays

A

Chromatin can be digested with nucleases and then resolved on agarose gel.
Gene-specific probes are used to find which genes are in the chromatin.
Can also be used to find nucleosome density

36
Q

How can histone modifications be revealed?

A

ChIP-seq:
Formaldehyde crosslinks DNA and proteins
DNA is fragmented and purified to find the sequence of interest
Then sequenced with third generation sequencing

37
Q

Where abouts in a gene is H3K9ac found most?

A

Transcription start sites (TSS)
This is where nucleosome structure is disrupted

38
Q

How were split genes first discovered?

A

Pulse Chase labelling showed that most labelled nuclear RNA (hnRNA) was degraded to a shorter, stable RNA rapidly after synthesis

39
Q

Outline pre-mRNA splicing

A
  1. 5’ exon is released from the transcript
  2. The two exons are joined together and the intron lariat is released
  3. Debranching enzyme makes it a linear RNA and exoribonucleases degrade the intron lariat
    Two transesterification reactions happen
40
Q

What are the 5 small nuclear ribonucleoproteins in the spliceosome complex?

A

U1 and U2 (RNA pol II)
U4, U5 and U6 (RNA pol III)

41
Q

What other complexes make up the spliceosome?

A

Prp19 allows catalysis of the reaction
ATPase Prp28 allows U4/U5/U6 to assemble on pre-mRNA
Snu114 and Brr2 induce U4/U6 unwinding
Prp16 and Prp22 allow exons to attach and intron to be removed

42
Q

Where do U1 and U2 bind on pre-mRNA?

A

U1: base pairs to the sequences 5’ end of the intron
U2: Branchpoint adenosine

43
Q

Outline spliceosome assembly

A
  1. U1 binds to the 5’ ssRNA splice site
  2. U2 binds to the branchpoint adenosine
  3. U4/U5/U6 (pol III) binds to U1 and U2
  4. U1 snRNP is released, followed by U4
  5. The Prp19 complex is recruited for catalysis
  6. U6/U2 catalyse splicing
44
Q

Outline kinetic proofreading for splicing

A
  1. Prp16 helicase is ATP driven. This facilitates the transition between the two transesterification reactions by lining up he exons
  2. If exons are misaligned after ATP hydrolysis, pre-mRNA is released and degraded
  3. With slower ATP hydrolysis, there is more time for exon alignment and can improve splicing success
45
Q

What is proposed to by the molecular clock of splicing?

A

Prp16: ATP driven helicase that lines up exons between the transesterification reaction

46
Q

How is splicing analysed in vitro?

A

RNAs are labelled with 32P and resolved by PAGE.
Looped RNA (intron lariat) moves slower than linear RNA due to a larger surface area for friction.

47
Q

What does the C-terminal domain (CTD) of RNA pol II contain?

A

Tandem repeats of hepatapeptide (YSPTSPS)
This allows spliceosome transcription to be coupled with RNA processing (capping/splicing/polyadenylation)

48
Q

Why can so many complexes bind to the C-terminal of RNA pol II?

A

It forms a rigid tail which gives a binding domain

49
Q

How are exon boundaries defined in pre-mRNA?

A

Proteins bridging U2 and U1 recognise protein-RNA and protein-protein interactions
-SR proteins mediate the interactions
-U2AF aids U2 binding to the branchpoint adenosine
-Exonic splicing enhancers (ESEs)

50
Q

What does alternative splicing involve?

A

Either a strong or weak canonical splice site
Enhancers or silencers
This can generate protein isoforms or regulate expression
Alternative splicing can exclude/include certain introns and exons

51
Q

Explain exon definition in the SMN2 gene

A

Gene loss of SMN2 underlies spinal muscular atrophy (SMA) which is muscle wasting.
The mutation has no effect on protein coding potential, but lies in an exonic splicing enhancer, blocking use of the 3’ splice site.

52
Q

Which gene is expressed by female flies in early embryogenesis?

A

Sex lethal (Sxl)
This is a splice site repressor
Expressed in a positive feedback loop

53
Q

What other gene does Sxl promote expression of?

A

Transformer (Tra) is expressed by exon exclusion
This is a splice site activator that regulates expression of doublesex (Dsx) from exon 4 with SR protein

54
Q

How does Sxl repress splice site binding?

A

Sxl contains two adjacent RRMs which have an affinity for U-rich and G/U-rich silencing elements
It binds to the 3’ ends of introns in the sxl and tra mRNAs.
RRMs also mediate actions with U2 to prevent it recognising the branchpoint adenosine

55
Q

What is trans-splicing?

A

The spliceosome uses the 5 splice site from one RNA and branchpoint A and 3’ splice site from another.

56
Q

Outline the minor “atac” spliceosome?

A

Some introns in eukaryotes have a non-canonical 5’ and 3’ splice site. These are removed by variants of U1 (U11), U2 (U12), U4 (atac) and U6 (atac)
U5 is the same in major and minor spliceosome

57
Q

What are group II introns?

A

Self-splicing introns which splice using the same mechanism as pre-mRNA splicing.

58
Q

What are group I introns?

A

Self-splicing introns which depend on a guanosine cofactor to attack the 5’ end of the intron
The splice site positions are mediated by snRNPs

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