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 binds to complementary strands to form double-stranded DNA.

Question 2

Single Stranded DNA

Answer 2

5’ (5 prime) and 3’ (3 prime) are numbered based on the carbon atoms of the sugar. The base is attached to the first carbon, the phosphate links between the 3’ of one sugar and the 5’ of the adjacent sugar. Numbering starts at the carbon closest to the base.

By convention single-stranded DNA sequence is listed 5’ to 3’.

Question 3

How does DNA exist in 3D?

Answer 3

• There are two antiparallel strands of DNA
• The bases are ‘stacked’
• There are two grooves: major and minor

Question 4

Genome variation
(interesting facts tbh)

Answer 4

• Human genome is 3x10’9 base pairs.
• It contains 20,000 genes.
• There is trend for simpler organisms to have fewer genes e.g. - flies (10,000), yeast (4,000), bacteria (1,000)

There is 2m of DNA in a nucleated cell, there are 37.2 trillion cells in your body so that’s 7.44x10’13 metres of DNA. = 250 journeys to the sun and back.

Question 5

How do we pack so much DNA into cells?

Answer 5

They’re packed into HISTONES.

• Basic (positively charged) proteins that bind DNA.
• Eight histones 2x(H2A+H2B+H3+H4) form the nucleosome.
• Histone 1 binds the linker DNA.

Question 6

What is the order in which DNA is packed?

Answer 6

• DNA basic helix
• nucleosomes
• chromatin fibres
• extended section of chromosome
• loops of chromatin fibre
• chromosome

Question 7

What are the 3 chromosome structures?

Answer 7

Metacentric - long and short arm of equal length.
Submetacentric - a long long arm and short short arm
Acrocentric - Long long arm and no short arm.

Centromere - gives orientation point.
Telomeres - ends of chromosome.

Human karyotype - an individual’s collection of chromosomes.

Question 8

What is the relation between DNA and genes?

Answer 8

Genome = 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 9

What is the exome?

Answer 9

The exome is made up of gene sequences. The exome is the part of the genome composed of exons, the sequences which, when transcribed, remain within the mature RNA after introns are removed by RNA splicing and contribute to the final protein product encoded by that gene.

Some definitions use all of the coding sequences (1.2% of genome)

Some definitions use all of the gene sequences (2% of genome)

Question 10

What is a gene?

Answer 10

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. Promoter function - on/off, where, when, how much?

It is NOT just the bits that encode the final protein, regulation of the gene is very important.

Question 11

What are the intergenic regions (in the chromosome)?

Answer 11

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

Intergenic regions are 98% of the genome.

Question 12

How is the genome organised?

Answer 12

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

This:

• allows for coordinate gene regulation
• may just reflect evolutionary history

Question 13

What do genes consist of?

Answer 13

Genes consist of a promoter and a transcription unit. The transcription unit consists of extrons (there are n number of extrons) and introns (n-1 introns). Some exons have coding and non-coding regions. The promoter is at the 5’ prime end. There are transcription and translation initiators and terminators.

Question 14

Introns in genes

Answer 14

• Vary in number – from 0 to at least 311
• Vary in size - 30bp to 1Mbp
• Some introns contain other genes
• Introns are crucial because the protein repertoire or variety is greatly enhanced by alternative splicing in which introns take partly important roles. Alternative splicing is a controlled molecular mechanism producing multiple variant proteins from a single gene in a eukaryotic cell.

Question 15

What are TATA boxes and regulatory elements?

Answer 15

TATA boxes are needed to recruit general transcription factors and RNA polymerase.

Regulatory elements are needed to regulate recruitment of RNA polymerase.

Question 16

What does the promoter do?

Answer 16

• 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 17

What are some other Regulatory Regions?

Answer 17

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).

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

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

Question 18

What happens to mRNA in transcription with RNA polymerase II?

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

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

Question 19

Very briefly, describe the steps of transcription.

Answer 19

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

Transcription –> primary transcript (exons and introns) a.k.a pre-mRNA. —> Post-transcriptional modifications ——> forms mature mRNA.

Question 20

What are the post-transcriptional modifications of mRNA?

Answer 20

• Capping - capped at 5’ end.

Splicing - spliced introns removed.

Polyadenylations - polyadenylated at 3’ end, adding lots of As at the end.

Question 21

What is capping (5’cap)?

Answer 21

After 25-30 nucleotides are synthesised, a methylated cap is added to the 5’ end by three enzyme activities:

• RNA 5’-triphosphatase
• Guanylyltransferase
• N7G-methyltransferase

This is added to prevent digestion.

The first two activities are carried out by a bifunctional capping enzyme (CE).

RNA Polymerase II is also required.

Question 22

What is the process of splicing introns?

Answer 22

Splicing is removal of introns by spliceosome and by joining of exons. 150 proteins form the mature spliceosome.

Linkage 2’-5’ in the intron. The catalysing of the extrons splices the introns out, giving a ‘lariat’ structure.

Once spliced, proteins associated with the Exon Junction Complex
help recruit the Transcription Export Complex (TREX complex) allowing for the targeting mRNA for nuclear export in endoplasmic reticulum.

Question 23

What happens in polyadenylation?

Answer 23

There is the addition of the 3’ POLY A tail.

• 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 is alternative splicing?

Answer 24

Exons can be skipped or added, so variations of a protein (called isoforms) can be produced from the same gene.

Question 25

Explain the 3D genome structure and how has it been identified?

Answer 25

Most of the time, DNA is not organised into chromosomes. In somatic cells the nuclear DNA is arranged non-randomly. The organisation has been identified using Hi-C (detects genomic DNA sequences in close proximity) and high-throughput microscopy. It involves the CTCF protein and Cohesin protein complex as well as the transcription machinery.

Question 26

What are the compartments that chromosomal DNA is arranged in?

Answer 26

The genome can be separated into compartments:

• Compartment A - transcriptionally active with histone modifications.
• Compartment B - transcriptionally repressed with repressive histone modifications.

These are interspersed throughout the 3D sequence but the same compartment types are bought closer together in the 3D genome.

Question 27

What are Topologically-Associated Domains (TADs)?

Answer 27

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 28

What is a method of transcriptional control?

Answer 28

The CTCF/Cohesin-regulated

Chromatin Loop Extrusion. The enhancer and promoter are also involved here. - Check slides.

Question 29

Describe pseudogenes.

Answer 29

These are genes that have been at least partially inactivated by the loss or gain of sequence that disrupts their correct transcription and/or translation.

Processed pseudogenes have no promoter or exons as they are copied from mRNA by retrotransposition.