W1 - Genome Structure

Question 1

What is 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

Question 2

What does a single strand of DNA consist of?

Answer 2

5’ and 3’ carbons are indicated – numbering starts at the carbon
closest to the base.
* Sequence is 5’->3’ by convention

Question 3

What does a double stranded DNA consist of?

Answer 3

Antiparallel, complementary base pairing.
The 5’ and 3’ go in diagonal and opposite directions making it antiparallel.

Question 4

How can DNA be seen in a 3D format?

Answer 4

• Two antiparallel strands of DNA
• Bases “stacked”
• Two grooves
• Major
• Minor

Question 5

How is there genome variation?

Answer 5

• Human genome is 3 x 10^9 base pairs – 3Gbp
• It contains ~20 000 genes.
• There is a trend for simpler organisms to have fewer genes,
  for example, flies have 10,000, yeast 4,000, bacteria 1,000
• However, genome size is not strongly related to complexity of an organism - marbled lungfish is 130Gbp and Paris japonica is 149Gbp

Question 6

What is the solution to the DNA packing problem?

Answer 6

Nuclear DNA = 2m per cell

Solution to packing:
* Basic (+vely charged) proteins that binds the negatively charged DNA
* Eight histones 2x(H2A+H2B+H3+H4) form the nucleosome
* Histone 1 binds the linker DNA

Question 7

What is the nucleosome structure with the DNA packed?

Answer 7

After being packed into histones, the DNA then wound around themselves to form fibres. They then wound further to form an extended chromosome. This is again wound to form loops of chromatin fibre to then produce the metaphase chromosome - the densest form of DNA.

Question 8

What is the definition of an exome?

Answer 8

• The exome is made up of gene sequences
• 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 9

What is a gene?

Answer 9

• 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.

Question 10

What is the structure of a gene?

Answer 10

Structure of chromosome is: Genes, Intergenic region (98%), Genes.

These genes can be very different sizes.
Intergenic regions contain sequences of unknown function, such as repetitive DNA, endogenous retroviruses, pseudogenes. They may contain many regulatory elements.

Genes often cluster in families – e.g. globin clusters:
- allows for co-ordinated gene regulation
- may just reflect evolutionary history

Structure of a gene: Promotor (CAATboxes and TATA boxes) and within the transcriptional unit, multiple exons and introns. Transcriptional initiation and termination. There are also 5’ UTR and 3’ UTR (untranslated region).

Question 11

What are introns and what is their purpose?

Answer 11

• Vary in number – from 0 to over 300
• Vary in size - 30bp to 1Mbp
• Some introns contain other genes

There is always one less intron than exons - none exist after final exon.

Purpose: Allow for splicing - multiple different forms of the same gene.

They do not exist in the matured RNA

Question 12

What is a promoter?

Answer 12

• 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 13

What is a regulatory element?

Answer 13

Regulatory element
(needed to regulate recruitment
of RNA polymerase)

Question 14

What is a TATA box?

Answer 14

“TATA box”
(needed to recruit general
transcription factors and RNA
polymerase)

Question 15

What are enhancers?

Answer 15

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 16

What are silencers?

Answer 16

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

Question 17

What are insulators?

Answer 17

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

Question 18

What is transcription?

Answer 18

• 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 19

What are the detailed stages of transcription?

Answer 19

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

1) The promotor and transcription unit exists.
2) RNA polymerase comes in and unwinds local DNA helix
3) RNA synthesis begins by unwinding from the 5’ to 3’ direction
4) Elongation
5) Termination
6) RNA polymerase dissociates with the pre-mRNA made.

Question 20

What is the post-translational modification of DNA?

Answer 20

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

Question 21

What is the 5’ cap?

Answer 21

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

Question 22

How are introns spliced?

Answer 22

This is done through a spliceosome, which loops out the intron and connects the two ends of the exon.
2’-5’ linkage is made in a lariat structure.

Question 23

What is the 3’ Poly A tail?

Answer 23

• 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

Question 24

What are the steps of translation?

Answer 24

The mRNA will be transported to a ribosome. The codons on the mRNA will code for anticodons carried by tRNA. Each different tRNA is covalently linked to an amino acid.

AUG (methionine) initiate translation - aka start codon. When this binds to the mRNA within the ribosome, this creates an initiation complex. Another tRNA will come to join the adjacent codon and the first will leave. This continues until a stop codon is reached.

Question 25

What is alternative splicing and how is it useful?

Answer 25

Exons can be skipped or added, so many variations of a protein (called isoforms) can be produced from the same gene. Proteins created are very complex as a result.

Question 26

What is the 3D genome structure?

Answer 26

* 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 27

What are the different compartments of a 3D DNA structure?

Answer 27

* The genome can be separated into 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 28

What are the Topologically Associated Domains (TADs)?

Answer 28

Topologically means close together in 3D space. * 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 29

What is 3D transcription control (Loop extrusion model of control of transcription)?

Answer 29

We have DNA bound by CTCF on either end of it. A Cohesin Complex comes in and pulls the DNA through itself - this is loop extrusion. The loop extrusion model enables the DNA to bring together an enhancer and promoter sequence - pulled together from the primary structure for them to interact in 3D space.