Genome structure

Question 1

Describe 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 (the double helix)

Question 2

Describe typical nucleotide structure

Answer 2

-phosphate group
-sugar (deoxyribose sugar or ribose sugar)
-nitrogenous base (A+T/U and G+C)

Question 3

Explain single stranded DNA

Answer 3

5’ and 3’ carbons are indicated- numbering starts at the carbon closest to the base
-the sequence is 5’-3’ by convention

Question 4

Explain DNA in 3D

Answer 4

-the double helix are two antiparallel strands of DNA
-sugar phosphate backbone with bases ‘stacked’ on the inside of the helix.

-there is a twisted shape with two grooves;
* major
* minor

Question 5

Discuss histones and the role of histones

Answer 5

-histones are positively charged (basic) proteins that bind DNA
-the key functions of histones: DNA packaging, structural support, gene regulation

-there are eight histones;
* 2x (H2A + H2B + H3 + H4) to form the nucleosome

Question 6

Explain the chromatosome

Answer 6

-Histone H1 binds to the DNA’s entry and exit points on the surface of the nucleosomal core particle
-histone H1 binding increases DNA compaction—-> aiding in the folding of chromatin into higher-order structure

Question 7

Discuss DNA packaging

Answer 7

DNA double helix—> nucleosomes—-> chromatin fibres—-> extended section of chromosome—-> loops of chromatin fibre——> metaphase chromosome

Question 8

Explain the genome vs the exome

Answer 8

The genome:
-is a complete set of an organism’s genetic material. Includes all DNA (both genes and non coding DNA)
-stores, propagates and expresses genetic information that guides cellular processes

The exome:
-the exome is the entire set of exons in an individual genome
-provides the genetic instructors for how to make proteins

Question 9

What is a gene?

Answer 9

-a gene contains a gene promotor, coding sequences and a stop codon.
DNA—-> RNA——> Protein

-all of the DNA that is transcribed into RNA, plus all of the local control regions are required to ensure quantitatively appropriate tissue- specific expression of the final protein

Question 10

Explain gene structure in terms of regions and sequences

Answer 10

-Intergenic regions (98% of the genome) may contain regulatory elements
-they also contain sequences of no known function, such as repetitive DNA, endogenous retroviruses, pseudogenes…

Question 11

Explain benefits of genes in cluster families (globin clusters)

Answer 11

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

Question 12

Explain introns in genes

Answer 12

-vary in number (from 0 to over 300)
-vary in size- 30bp to 1Mbp
-some introns contain other genes
-some genes have no introns, such as histones

Question 13

Discuss the promotor

Answer 13

The promoter is known as the TATA box which is needed to recruit general transcription factors and RNA polymerase

-the regulatory elements is needed to regulate recruitment of RNA polymerase

Question 14

List and explain some other regulatory regions

Answer 14

-enhancers upregulate gene expression—-> they are short sequences that can be in the gene or many kilobases distant. They are targets for activators

-Silencers downregulate gene expression—-> they are also position-independent and are targets for depressors

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

Question 15

Explain transcription

Answer 15

-messenger RNA synthesis (transcription) is catalysed by RNA polymerase II
-transcribes in 5’ to 3’ direction
-transcribes everything after the transcription start site
-mRNA is post transcriptionally modified

-Transcription is a biological process where the genetic code of a DNA segment is copied into mRNA
-RNA polymerase II recognises promoters efficiently with the assistance of many other transcription factors

Question 16

Explain the transcription unit

Answer 16

-RNA polymerase recruited (closed complex)
-DNA helix locally unwound (open complex)
-RNA synthesis begins
-Elongation
-Termination
-RNA polymerase dissociates.

Question 17

Explain post-transcriptional modification of mRNA: the 5’ cap

Answer 17

-After 25-30 bases are added, 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)
-capping occurs during transcription in the nucleus and RNA polymerase II is also required
-functions: protection, nuclear export, translation, splicing

Question 18

Explain post transcriptional modification of mRNA: Splicing of introns

Answer 18

-performed by a complex molecular machine, the spliceosome is roughly 150 proteins that assembles at the boundaries between introns and exons
-it recognises the start and end of each intron, cuts it out of the pre-mRNA and releases it in a loop-like structure

-the two flanking exons are ligated together

Question 19

Explain the importance of splicing of introns

Answer 19

-proteins synthesis; splicing ensures that only protein-coding sequences are translated

-alternative splicing: a single gene can produce multiple different proteins where different combinations of exons are included in the final mRNA.

Question 20

Explain alternative splicing and protein diversity

Answer 20

-by alternative splicing, exons can be skipped or included
-this leads to many variants of a protein (isoforms) from a single gene increasing protein diversity

The physiological significance; embryo development, tissue differentiation.

Question 21

Explain the post transcriptional modification of mRNA; 3’ poly A tail

Answer 21

-the 3’ Poly (A) tail is 100-250 adenine nucleotides sequence added to the 3’ end of mRNA molecules during post-translational processing
-as transcription of a gene terminates, pre-MRNA is cleaved and a poly A tail is synthesised by complex proteins
-contributes to mRNA stability, nuclear export and translation efficiency

Question 22

Explain the roles of CPSF and PAP

Answer 22

CPSF (cleavage and polyadenylation stimulating factor) recognises the PAS (polyadenylation signal) and acts on the cleavage site. CSTF (cleavage stimulation factor) recognises GU-rich downstream elements (DSE)

-PAP (poly A-polymerase) is recruited and adds multiple adenine bases after cleavage site. Other proteins are required for this process- PAB (poly-A binding protein), CFLm (cleavage factor Im), CFllm and Simplekin

Question 23

Explain the key characteristics of translation

Answer 23

-key components; mature mRNA, the ribosome, tRNA and amino acids
-three stages: initiation, elongation and termination (stop codons UAA, UAG, or UGA)

Question 24

Explain the 3D genome structure

Answer 24

-most of the time DNA is not organised into chromosomes
-in somatic cells the nuclear DNA is arranged non-randomly
-organisation has been determined using Hi-C (detects genomic DNA sequences in close proximity) and high- throughput microscopy.

-involves the CTCF protein and cohesion protein complex, as well as the transcription machinery to create stable DNA loops

-stable DNA loops formed by the cooperation of CTCF and cohesion define topologically associating domains (TADs)

Question 25

Explain DNA compartments

Answer 25

-the genome can be separated into two compartments: * compartment A- transcriptionally active with activating histone modifications * compartment B- transcriptionally repressed with repressive histone modifications -these are interspersed throughout the 2D sequence -the same compartment types are brought closer together in 3D space.

Question 26

Explain topologically-associated domains (TADs)

Answer 26

-individual compartments are made up of several non-interacting sub-compartments -TADs are self interacting regions of the genome where DNA sequences within a TAD interact more frequently with eachother than with sequences outside the domain -TADs regulate gene expression by co-localising regulatory elements with their target genes -they are usually separated by the transcriptional repressor CTCF protein

Question 27

Explain TADs and 3D transcription control

Answer 27

-mechanism by which CTCF/ Cohesin regulated chromatin loop extrusion can contribute to the formation of enhancer-promoter interactions. 1) Cohesin Complex (the blue rings) – works like a “sliding clamp” that pulls DNA through to form loops. 2) CTCF proteins (orange flowers) – act like “roadblocks” that stop cohesin at specific sites. 3) Loop Extrusion – cohesin loads onto DNA, starts pulling it through, and creates loops until it hits CTCF. 4) Enhancer-Promoter Interactions – loops bring enhancers (yellow dots) close to promoters (purple dots), allowing genes to be switched on more efficiently. 5) Outcome – 3D looping makes sure the right enhancers connect to the right promoters, helping regulate which genes are expressed.

Question 28

Explain regions of chromosomes relationship with colour

Answer 28

-regions of the chromosomes where genes are active and coloured blue, and the regions that interact with the nuclear lamina (a dense fibrillar network inside the nucleus) are coloured yellow