Genome structure

Question 1

In its 3D structure why does DNA have a major and a minor groove?

Answer 1

Because the glycosidic bonds that bond the base pairs to the sugar phosphate backbone aren’t directly opposite each other

Question 2

Describe the process of DNA supercoiling

Answer 2

• It occurs when the DNA double helix is wound around 8 histone proteins (an octamer of histones)
• The octamer of histones includes 2 molecules each of the 4 different types of histone protein: Histone 2A, 2B, 3 and 4.
• This coiling of DNA around the histone proteins forms a nucleosome

Question 3

Describe the role of histone 1 in DNA supercoiling once the nucleosome has been formed

Answer 3

Histone 1 binds to the outside of the nucleosome and then to the linker DNA. This binding neutralises the negative charge on the linker DNA allowing it to link nucleosomes together.

Question 4

Formation of the nucleosome is the first level of DNA packaging. Describe the the steps involved in the other levels of DNA packaing which result in the formation of the chromosome.

Answer 4

1. Nucleosomes bound together via histone 1 and which causes the formation of the 30nm fibre (Solenoid fibre)
2. Solenoid fibre coiled and looped further via addition of scaffold proteins to form 80-100nm fibre
3. Further scaffold proteins added to 80-100nm fibers causing them to be packaged further into a chromosomal-like structure
4. Chromosomal-like structure is condensed even further to form loops of chromatin
5. Loops of chromatin can be condensed to form chromosomes

Question 5

What form is DNA usually present in within the nucleus of a cell?

Answer 5

DNA normally seen as a structure that resembles the loops of chromatin.

Question 6

When is DNA in the nucleus present in the form of chromosomes?

Answer 6

When a cell undergoes mitosis/meiosis

Question 7

What are the 3 types of chromosome structure?

Answer 7

• Metacentric - Centromere is in the middle of the chromosome
• Submetacentric - Centromere positioned closer to one end of the chromosome
• Acrocentric - Chromosome has no short arms

Question 8

What is genome size and is it correlated to organism complexity? E.g. A more complex organism will have a larger genome size than a less complex one

Answer 8

• Genome size is defined as the total amount of DNA contained within one copy of a single complete genome.
• Genome size is not really related to organism complexity as the human genome size is 3 x 109 base pairs – 3Gbp. There are less complex organisms that have a larger genome size e.g. marbled lungfish has a genome size of 130Gbp

Question 9

Is gene number more closely correlated to organism complexity than genome size is?

Answer 9

Yes gene number is more closely correlated to organism complexity as the trend tends to be the simplier organisms have fewer genes. E.g. humans have around 20,000 genes while bacteria only have around 1,000 genes.

Question 10

What are the 2 different definitions of the exome?

Answer 10

• Exome is only the coding sequence

Exome is the whole gene sequence (includes regulatory signals and parts of gene sequence with no function)

Question 11

What is the definition of a gene?

Answer 11

All of the DNA that is transcribed into RNA plus all of the local control regions that are required to quantitatively regulate tissue-specific expression of the gene

Question 12

Within the genome why is it that genes tend to cluster into families?

Answer 12

• Allows for coordinated gene regulation - Cluster of genes may make up part of macromolecule so ideal for them to be close together and be regulated by the same signals
• May just reflect evolutionary history of those genes

Question 13

The regions between genes are called intergenic regions. What structures are thought to be found within these intergenic regions?

Answer 13

• Repetitive DNA
• Endogenous retroviruses (no longer functional)
• Pseudogenes (genes that have lost at least some functionality).
• May also contain many regulatory elements.

Question 14

What are the major strcutures found within a gene?

Answer 14

• Promoter region - controls the expression of the gene by allowing RNA polymerase to bind
• Exons (situated within the transcription unit) - the part of the DNA that is transcribed into RNA
• Introns (Between the exons) - These get removed from the pre-mRNA to form mature mRNA

Question 15

What other important structures do you find within a gene?

Answer 15

• DNA sequences that promote gene expression e.g. TATA box - found within the promoter region
• Transcription initiation site
• Translation initiation site
• Translation termination site
• Transcription termination site

Question 16

Why is it that an exon isn’t necessarily classed as a coding sequence?

Answer 16

• Because the 5’ untranslated region (UTR) is a part of the first exon within a gene whilst the 3’ untranslated region is part of the last exon.
• Untranslated regions don’t get translated into protein and so don’ code for anything
• In fact an entire exon may be made up of an untranslated regions.

Question 17

What is a promoter region and what is its primary function?

Answer 17

• The sequence immediately 5’ to the region to be transcribed is called a “promoter”
• Promoters recruit RNA polymerase to a DNA template

Question 18

How is RNA polymerase recruited to the promoter region?

Answer 18

• TATA box recognised and bound to by TATA binding protein (TBP) found within TFIID subunit of TFII basal machinary
• Rest of TFII basal machinary recruited to form complete pre-initiation complex
• Pre-initiation complex then allows for RNA polymerase to bind to the promoter region

Question 19

What is an enhancer?

Answer 19

Short sequences of DNA that act to enhance the recruitment of RNA polymerase to a promoter

Question 20

What does it mean when it is said that enhancers work in a “postion independent manner?”

Answer 20

It means that an enhancer can be located in the gene, near the gene or far away from the gene and still exhibit its effects on that gene.

Also, means that the enhancer can be located either 5’ or 3’ of the gene and still work

Question 21

What does it mean when it is said that an enhancer works in an “orientation independent manner?”

Answer 21

This means that enhancers will still work if put in the opposite orientation, e.g. If the original orientation is 5’ to 3’ and you place the enhancer 3’ to 5’ then it’ll still work.

Question 22

What is a silencer?

Answer 22

Short sequences of DNA that downregulate gene expression by making recruitment of RNA polymerase to the promoter more difficult.

Question 23

What is an insulator?

Answer 23

Short sequences that act to prevent enhancers/silencers influencing genes other than their target genes

Question 24

Do silencers and insulators also work in orientation and position independent manners?

Answer 24

Yes

Question 25

What are the 3 processes that cause pre-mRNA to become mature mRNA?

Answer 25

* Capping * Polyadenylation * Splicing

Question 26

Explain the process of capping

Answer 26

* Shortly after beginning transcription, a guanine nucleotide is added to the 5’ end of the mRNA as well as a 5'-5’ triphosphate bridge. * The guanine nucleotide that was added to the 5' end of the mRNA is then methylated at position 7 by the enzyme methyltransferase to give rise to the 7 methylguanine cap.

Question 27

What other positions on the mRNA can be methylated during capping?

Answer 27

The final 2 bases of the mRNA can also be methylated by the methyltransferase enzyme

Question 28

Explain the process of polyadenylation?

Answer 28

* After the stop codon there is a sequence called polyadenylation signal or termination signal (AAUAAA). * This sequence is recognised by the CPSF (cleavage and polyadenylation specificity factor) which then cleaves the DNA after the polyadenylation signal. * Polyadenylate polymerase then adds 250 adenosine monophosphates to the 3’ end of pre-mRNA to produce the poly A tail.

Question 29

Explain the process of splicing

Answer 29

* snRNPs (small nuclear ribonucleoproteins) bind to the introns of the pre-mRNA that contain GU at the 5’ splice site (5’ SS and AG at the 3’ SS. * Spliceosome then cleaves the GU at the 5’ SS of intron * GU sequence then loops around and joins with the branch point (A or adenine) to form lariat – like intermediate * Spliceosome then cleaves the AG at the 3’ SS of the intron * Ligase then joins the 2 exons together

Question 30

What is the role of the splice sites within the pre-mRNA?

Answer 30

Splice sites mark where the intron is within the pre-mRNA

Question 31

When the GU sequence joins the branch point (A) how is it that the guanine of the GU bonds with the adenosine of the branch point?

Answer 31

The bonding of the guanine and adenine takes place via transesterification in which a hydroxyl group (OH) on the carbon atom of the adenine ‘attacks’ the bond of the guanine nucleotide at the splice site

Question 32

Explain the idea of alternative splicing

Answer 32

Alternative splicing is the idea that not all the exons present within a gene are needed to produce mature mRNA which means that exons can be removed or even added to pre-mRNA to produce variations of a protein (called isoforms) from the same gene.

Question 33

What is a constitutive exon?

Answer 33

An exon that are always present within the mature mRNA

Question 34

What is an alternatively spliced exon?

Answer 34

An exon that can be removed from the pre-mRNA transcript.