Genome Structure

Question 1

Describe the basic structure of DNA

Answer 1

• DNA is deoxyribonucleic acid
• It is a macromolecule consisting of a linear strand of nucleotides
• Single linear strands bind to complementary strands to form double-stranded DNA
Two antiparallel strands of DNA
Bases “stacked”
Two grooves
• Major
• Minor

Question 2

Describe the charge of a DNA molecule

Answer 2

Negatively charged because of the phosphates

Question 3

What type of molecule is a single strand of DNA

Answer 3

linear macromolecule

Question 4

How many base pairs is the human genome and how many genes are there in a human?

Answer 4

• Human genome is 3 x 109 base pairs – 3Gbp

* It contains ~20 000 genes.

Question 5

What is the main issue with DNA?

Answer 5

DNA is very lengthy:

There is around 2m of DNA in a nucleated cell
• 37.2 trillion cells in your body
• That is 7.44x1013 metres of DNA

Question 6

So how do we fit DNA into nucleated cells?

Answer 6

The solution is Histones

Question 7

Describe the charge of histones

How many histones form a nucleosome?

What binds the linker DNA

Answer 7

Basic positively charged that binds to negative DNA

Eight histones 2x(H2A+H2B+H3+H4) form the nucleosome

Histone 1

Question 8

Have a look at the image of X-ray crystallography of DNA around histones

Answer 8

On image

Question 9

What are the phases of DNA packing?

Answer 9

1. DNA double helix
2. Nucleosomes
3. Chromatin fibre
4. Extended section of chromosome
5. Loops of chromatin fibre
6. Metaphase chromosome

Question 10

Descirbe the structure of a chromosome

Answer 10

On image

Question 11

What is a human karotype and what does it show?

Answer 11

• Stained chromosomes, nucleus in metaphase

* Banding patterns

Question 12

Define genome (3)

Answer 12

• The primary DNA sequence encodes all the gene products necessary for a human
• The primary DNA sequence also includes a large number of regulatory signals
• Much of the DNA sequence does not have an assigned function as yet

Question 13

Define exon (there are two)

Answer 13

• The exome is made up of gene sequences
Coding regions of DNA
• Some definitions use all of the coding sequences (~37 Mbp – 1.2% of genome)
• Some definitions use all of the gene sequences (~60Mbp – 2% of genome)

Question 14

Define gene and describe the structure of a gene

Answer 14

• All of the DNA that is transcribed into RNA plus all of the cis-linked (local) control regions that are required to ensure quantitatively appropriate tissue-specific expression of the final protein
• It is NOT just the bits that encode the final protein, regulation of the gene is very important.

On image

Question 15

Where are the size of the genes globin and dystrophin ?

Answer 15

globin = 1.8kb, dystrophin = 2.4Mb

Question 16

What are intergenic regions?

Answer 16

Intergenic regions contain sequences of no known function, such as repetitive DNA, endogenous retroviruses, pseudogenes. They may contain many regulatory elements.

Question 17

How do genes tend to exist and what does this allow?

Answer 17

Genes often cluster in families – e.g. globin clusters

• allows for co-ordinated gene regulation
• may just reflect evolutionary history

Question 18

What is an intron?

How many are there and what is the size of them?

Answer 18

Non-coding regions of DNA

• Vary in number – from 0 to at least 311
• Vary in size - 30bp to 1Mbp

Question 19

Describe the structure of a gene

Answer 19

On image

Question 20

What are the two major regions that the promoter region contains and what are there functions?

Answer 20

1. Regulatory region - needed to regulate the recruitment of RNA polymerase 2
2. TATA box - needed to recruit general transcription factors and RNA polymerase

Question 21

3 general functions of the promoter region

Answer 21

• Promoters recruit RNA polymerase to a DNA template
• RNA polymerase binds asymmetrically and can only move 5’ to 3’
• Regulation occurs via transcription factors

Question 22

What are the purpose of enhancers?

Answer 22

• Enhancers upregulate gene expression – they are short sequences that can be in the gene or many kilobases distant. They are targets for transcription factors (activators).

Question 23

What are the purpose of silencers?

Answer 23

• Silencers downregulate gene expression. They are also position-independent and are also targets for transcription factors (repressors).

Question 24

What are insulators?

Answer 24

• Insulators are short sequences that act to prevent enhancers/silencers influencing other genes

Question 25

What is transcription catalysed by? In what direction does it catalyse the reaction? Does it trascribe coding AND non-coding regions?

Answer 25

* Messenger RNA synthesis (transcription) is catalysed by RNA Polymerase II * Transcribes in 5’ to 3’ direction * Transcribes everything after the transcription start site (exons and introns) * mRNA is post-transcriptionally modified

Question 26

What is the purpose of RNA polymerase 2 and look at its structure?

Answer 26

RNA polymerase II recognises promoters efficiently with the assistance of many other transcription factors

Question 27

What are the 7 stages of transcription?

Answer 27

1. DNA 2. RNA polymerase recruited (closed complex) 3. DNA helix is locally unwound (open complex) 3. RNA synthesis begins 4. Elongation 5. Termination 6. RNA polymerase dissociates

Question 28

What does transcription produce? What does post-transcriptional modification produce?

Answer 28

pre-mRNA mRNA

Question 29

What 3 process form post-transcriptional modification?

Answer 29

* Capped at 5’ end * Spliced - introns removed * Polyadenylated at 3’ end

Question 30

Describe how a 5' capped is formed? Whats the purpose of the 5' cap?

Answer 30

After 25-30nts are synthesised, a methylated cap is added to the 5’ end by three enzyme activities: • RNA 5’-triphosphatase • Guanylyltransferase • N7G-methyltransferase The first two activities are carried out by a bifunctional capping enzyme (CE) RNA Pol II is also required Makes it resistant to digestion by enzymes within the cell

Question 31

How is the 3' Poly A tail formed? What's the purpose?

Answer 31

CPSF (Cleavage and Polyadenylation Stimulating Factor) recognises the PAS (Polyadenylation signal) and acts on cleavage site • CSTF (Cleavage Stimulating Factor) recognises GU-rich Downstream Elements (DSE) • PAP (Poly-A polymerase) is recruited and adds multiple A bases after cleavage site • PAB is Poly-A Binding Protein. Other proteins appear to be required for this process – CFIm (Cleavage Factor Im), CFIIm and Simplekin - The 3’ poly A tail o Around 250 As are added o Protects the end from degradation o Also help target messages out of the nucleus

Question 32

Describe the process of splicing What is it? What catalyses it? What happens in the process?

Answer 32

Splicing of introns (removal of introns) • The spliceosome catalyses this reaction • The spliceosome (150 proteins) catalyses the OH group to the P group • This frees up the Oh group, linked to phosphate • 2’ to 5’ linkage • Its then spliced out and is known as the lariat

Question 33

What happens after splicing?

Answer 33

splicing targets mRNAs for nuclear export: Once they are spliced, TRanscription-Export (recruited by Exon Junction Complex) which helps to export this mRNA out of the nucleus. This allows targeting of the MRNA to the ER.

Question 34

Give an outline of the whole process

Answer 34

On image

Question 35

What is Alternative splicing?

Answer 35

- We can choose to splice out different exons as well as introns and so can produce new proteins as a result. - We can skip or even add exons to produce variations of a protein from the same gene (isoforms)

Question 36

How do we get protein variation?

Answer 36

On image

Question 37

How is DNA organised in somatic cells and how is it identified? What did it involve and require?

Answer 37

* In somatic cells the nuclear DNA is arranged non-randomly * Organisation has been identified using Hi-C (detects genomic DNA sequences in close proximity) and high-throughput microscopy * Involves CTCF protein and Cohesin protein complex, as well as transcription machinery

Question 38

What are the 2 compartments that the genome is found in?

Answer 38

* Compartment A – transcriptionally active with active histone modifications * Compartment B – transcriptionally repressed with repressive histone modifications These are interspersed throughout the 2D sequence but the same compartment types are brought close together in the 3D genome

Question 39

What are Topologically-Associated Domains (TADs)? | What are they separated by?

Answer 39

* Individual compartments are made up of several non-interacting sub-compartments * These are Topologically-Associated Domains (TADs) * They are usually separated by the Transcriptional Repressor CTCF protein

Question 40

Have a look at transcriptional control and genome structure (3D)

Answer 40

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