Organisation & Control of Eukaryotic Genome

Question 1

Packing of DNA in euk chromosomes

Answer 1

• negatively-charged double stranded DNA wound around octamer of histone proteins which are positively charged , forming nucleosome which are joined by linker DNA
• further folding and coiling of nucleosomes and linker DNA results in the solenoid structure, which appears as 30nm chromatin fibre
• further folding of the 30nm fibres into loops attached to non-histone protein, forming looped domains
• 300nm fibres are further folded to form one of the two chromatids observed during metaphase, also known as 700nm chromatin fibres

Question 2

Promoter

Answer 2

• non-coding sequences comprising of TATA box which allows TATA binding proteins to bind to it
• recruits general transcription factor and RNA polymerase for initiation of transcription

Question 3

Enhancer

Answer 3

• non-coding sequences which activators bind to
• increases interaction between RNA polymerase and promoter, hence increasing rate of transcription
• occurs by the looping of DNA, which further stabilises the transcription initiation complex through protein-protein interaction

Question 4

Why is three different temperatures required during one PCR cycle

Answer 4

95 - denaturation of dsDNA into ssDNA by breaking the hydrogen bonds between the nitrogenous bases of the two strands
55 - primers form hydrogen bonding, to the flanking sequence of the target DNA sequence due to CBP to the ssDNA
70 - optimum temperature for taq polymerase to add nucleotides to the 3’ OH end of the primers using ssDNA as template

Question 5

Outline the main principles that allow gel electrophoresis to separate DNA fragments

Answer 5

• DNA fragments pass through a porous gel which functions as a molecular sieve in an electric field
• separating DNA fragments by size where larger molecules move at a slower rate while smaller molecules move at a faster rate
• since DNA fragments are negatively charged, DNA fragments move from the negative electrode go to the positive electrode

Question 6

Functions of telomerase

Answer 6

• prevent ends of chromosome from being degraded by exonucleases
• protect important genes near the ends of telomerase by delaying the degradation of genes
• prevent ends of chromosomes from accidentally fusing with each other
• provide a counting mechanism for the number of cell divisions a cell has undergone

Question 7

DNA methylation

Answer 7

• addition of methyl groups to cytosine bases of DNA by methyl transferases results in methylated DNA, which recruits histone deacetylation enzymes
• catalyse the removal of acetyl groups from lysine residues in the tails of histone proteins, increasing the interaction between positively-charged histone proteins and negatively-charged DNA
• results in a more compact chromatin structure therefore less accessible to transcription machinery, reducing rate of transcription

Question 8

Histone modification

Answer 8

• histone acetyltransferases catalyse addition of acetyl groups to positively charged lysine residues in the tails of histone proteins
• positive charge is neutralised and reduces interaction between the histone and DNA
• make chromatin structure less compact and more accessible to transcription machinery, promoting transcription

Question 9

Transcription (in euk)

Answer 9

INITIATION:
- TATA binding proteins recognises the TATA box of the promoter and recruits general transcription factors and RNA polymerase to form transcription initiation complex
- DNA is unwound and separated by RNA polymerase for CBP of template strand to ribonucleotides
ELONGATION:
- RNA polymerase moves along the DNA and adds ribonucleotides to the free 3’ OH end of the growing RNA chain which is synthesised in the 5’ to 3’ direction via CBP with template strand
- RNA polymerase catalyse the formation of phosphodiester bonds between 3’ OH end of RNA and 5’ phosphate group of ribonucleotides added
TERMINATION:
- polyadenylation signal sequence (AAUAAA) is transcribed which causes termination to occur