Genome Structure

Question 1

What is the DNA?
What is it composed of?

Answer 1

• DNA is a deoxyribonucleic acid
• It is a macromolecule consisting of a linear strand of nucleotides
• The single linear strands bind to it’s complementary strands forming a double stranded DNA

Question 2

What is the Nucleotide made up of?

Answer 2

• A sugar molecule
• Nitrogenous base
• Phosphate group

Question 3

Describe the single stranded DNA

Answer 3

• The Nucleotides attach to eachother from the Carbon 3 of one molecule to the Carbon 5 of another molecule
• 5’ and 3’ carbons are indicated
• The sequence is 5’ -> 3’ by convention

Question 4

Describe the DNA in three dimensions

Answer 4

• Has 2 anti parallel strands of DNA
• The bases are stacked
• Has two grooves = Major/minor

Question 5

How big is the human genome?

Answer 5

The human genome is about 3 x 10^9 base pairs - 3Gbp

Question 6

How many genes does the human genome contain?

Answer 6

Contains about 20 000 genes

Question 7

Describe the trend in genes in regards to the human genome

Answer 7

• The simpler the organism, the fewer genes they have. For e.g. flies have 10 000, yeast have 4000, bacteria 1000
• However the genome size is not always strongly related to the complexity of the organism

Question 8

What is the problem with DNA in the body?

Answer 8

• The body has about 2 meters of DNA in a nucleated cell with about 37.2 trillion cells in the body.
• That’s about 7.44 x 10^13 meters of DNA
• The average cell is 50 ùm in diameter so how does the DNA fit

Question 9

How is this problem resolved?

Answer 9

• Solved through a type of protein called Histone proteins
• They’re basic positively charged proteins that bind DNA
• Eight histones 2x(H2A+H2B+H3+H4) form the nucleosome
• Histone 1 binds to the linker DNA

Question 10

Describe the Chromosome structure

Answer 10

• The metacentric has a short arm and long arm
• The Submetacentric has a centromere (At centre), Telomeres at the bottom ends and chromatids at the top ends
• The acrocentric has Satellites on the centromeres

Question 11

Describe the primary DNA sequence

Answer 11

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

Question 12

Describe the Exome

Answer 12

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

Question 13

What is a gene?

Answer 13

• The gene is all of the DNA that’s transcribed into RNA including the Cis linked regions
• Cis linked regions 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

Question 14

Describe the gene structure (View diagram on slide)

Answer 14

Has a 2 gene region at the start of the gene followed by the inter gene region followed by another gene region. (These genes can often be very different in size)

Question 15

Describe the interagency region

Answer 15

Intervened regions contain sequences which have no known functions such as repetitive DNA, endogenous retroviruses, pseudo genes. They can contain many regulatory elements.

Question 16

Gene Structure. Describe the composition of the gene itself.

Answer 16

Genes often cluster in families
- Allows for co-ordinated gene regulation
- May just reflect evolutionary history

Question 17

Describe the introns present in genes

Answer 17

• Vary in number (from 0 - 311)
• Vary in size (30bp - 1Mbp)
• Some introns contain other genes

Question 18

What is the role of introns in a gene?

Answer 18

Introns can increase transcription levels by affecting the rate of transcription, nuclear export, and transcript stability.

Question 19

Describe the promoter region

Answer 19

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

Question 20

Describe the Other Regulatory Regions - Enhancers

Answer 20

Enhancers up-regulate gene expression
- They’re short sequences that can be in the gene or many kilo bases distant.
- They’re targets for transcription factors (Activators)

Question 21

Describe the Other Regulatory Regions - Silencers

Answer 21

Silencers down-regulate gene expression.
- They’re position independent
- They’re also targets for transcription factors (repressors)

Question 22

Describe the Other Regulatory Regions - Insulators

Answer 22

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

Question 23

Describe Transcription - Messenger RNA synthesis

Answer 23

• Catalysed by RNA polymerase II
• Transcribes in the 5’ to 3’ direction
• Transcribes everything after the transcription start site
• mRNA is post transcriptionally modified

Question 24

What is the function of RNA polymerase II?

Answer 24

Recognises promoters efficiently with the assistance of many other transcription factors

Question 25

Outline the steps of Transcription

Answer 25

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

Question 26

What are the 3 types of Post Transcriptional Modifications?

Answer 26

Capping Splicing Polyadenylation

Question 27

Where does the Post-transcriptional modifications occur?

Answer 27

- Capping - at the 3’ end - Splicing - Introns removed - Polyadenylation - at the 3’ end

Question 28

Describe the 5’ Cap

Answer 28

After 25-30 nts are synthesised, a methylated cap is added to the 5’ end by 3 enzyme activities: - RNA 5’ - triphosphatase - Guanylyltransferase - N7G-methyltransferase The first 2 activities is carried out by a bifunctional capping enzyme. RNA Pol II is also required

Question 29

Describe the 3’ poly A tail

Answer 29

1 - CPSF (Cleavage and Polyadenylation Stimulating Factor) recognises the PAS (Polyadenylation signal) and acts on cleavage site 2 - CSTF (Cleavage Stimulating Factor) recognises GU-rich Downstream Elements (DSE) 3 - PAP (Poly-A polymerase) is recruited and adds multiple A bases after cleavage site 4 - PAB is Poly-A Binding Protein. Other proteins appear to be required for this process – CFIm (Cleavage Factor Im), CFIIm and Simpleki

Question 30

What is the point of Splicing?

Answer 30

Splicing makes genes more "modular," allowing new combinations of exons to be created during evolution.

Question 31

What is alternative splicing?

Answer 31

Where Exons are skipped or added so many variations of a protein can be produced from the same gene

Question 32

Describe the 3D genome structure

Answer 32

- Most of the time, DNA is not organised into chromosomes - 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 33

How are the Compartments of the genome organised?

Answer 33

The genome can be separated into 2 compartments - 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 34

Describe the TOPOLOGICALLY-ASSOCIATED DOMAINS (TADs)

Answer 34

- Individual compartments are made up of several non interacting sub compartments (TADs) - They’re usually separated by transcriptional repress or CTFC protein