The Increased Complexity Associated with Eukaryotes

Question 1

what is chromatin and where is it wound around

Answer 1

Chromatin is DNA wound around octamer cores of histone proteins (lysine and arginine) to form nucleosomes

Question 2

how does chromatin appear and becomes known as chromatin fibre

Answer 2

‘beads on a string’
organised into helical array- becomes known as chromatin fibre
6 nucleosomes per turn of its helical array
Nucleosomes attach to protein scaffold which condense into larger fold and eventually form chromosomes.

Question 3

Describe properties of histone protein

Answer 3

• basic
• hence positively charged
• bind to DNA at physiological PH

Question 4

Describe the role of the cell cycle

Answer 4

4 stage process
Gap 1
- Metabolic changes prepare the cell for division
-The cell becomes committed to division after restriction point (R)
Synthesis
- DNA replication produces a copy of the genetic material
- Each chromosome now consist of two sister chromatid
Gap 2
- Metabolic changes assemble cytoplasmic materials necessary for mitosis and cytokinesis
Mitosis
- nuclear division followed by cell division
G,S,G2 - interphase

Question 5

how is the cell cycle regulated

Answer 5

Restriction point (R) - located at G1 phase, ensures cell doesn’t replicate damaged DNA
G2- checkpoint - validates correct DNA replication

Question 6

what is the biological significance in DNA replication

Answer 6

• extreme accuracy is necessary to preserve integrity of the genome in successive generations
  -High fidelity in eukaryotes due to slower replication rates
• Eukaryotic chromosomes are larger and more complex than prokaryotic material, there they are replicated from multiple origins of replication
  -The enzymes involved in eukaryotic DNA replication are more complex that hose in prokaryotic DNA replication

Question 7

Summarise DNA replication

Answer 7

Helicase using energy from ATP hydrolysis opens up DNA helix.
replication forks are formed at each replication origin as the DNA unwinds.
2. the opening of the double helix causes over winding - resolved with topoisomerases
3. Primers are formed by the enzyme PRIMASE.
Using the primer DNA polymerase can start synthesis
4. 3 major DNA polymerases are then involved:
α, δ and ε.
a. DNA pol α adds a short (20 to 30 nucleotides) DNA fragment to the RNA primer on
both strands, and then hands off to a second polymerase
b. While the leading strand is continuously synthesized by the enzyme pol δ, the lagging
strand is synthesized by pol ε

5. A sliding clamp protein known as PCNA holds the polymerase in place so it doesn’t slide of DNA
6. At the end of DNA replication, the RNA primers are replaced by DNA- 5’-3’ exonuclease and polymerase activity of DNA polymerase ε
7. The okazaki fragments in the lagging strand are joined after the replacement of the primers with the DNA
8. The gaps that remain are sealed by DNA ligase which froms the phophsdiester bond.

Question 8

what is the difference between prokaryotic and eukaryotic replication

Answer 8

Prokaryotes
- single origin of replication
- replicate 1000 nucelotides/s
- 5 DNA polymerase types
- no telomerase
- use DNA pol I to remove RNA primer
- DNA pol III to elongate strand

Eukaryotes
- multiple origin of replication
-50-100 nucelotides rate of replication
- 14 DNA polymerase type
- Telomerase needed
- RNase H- RNA primer removal
-Pol alpha,delta,epsilon

Question 9

what is telomeres and what is the problem in eukaryotic DNA

Answer 9

Telomers - ends of chromosomes (protective caps)
Eukaryotic DNA has issue with replicating telomers

-synthesis is difficult in lagging strand bc there is no primer possible for the end of the strand. Therefore this DNA would remain unpaired and chromosmes get shorter when cell divides

Question 10

what is telomerase

Answer 10

• enzyme that produces telomeres
• Contains RNA region so can be used as template
• extend 3’ lagging stand to allow replication of chromosomes to complete
• occurs as binding-polymerisation translocation

Question 11

what sequence is repeated in telomere regions

Answer 11

TTAGGG
TTGGGG

Question 12

what does telomerase contain and how does it work

Answer 12

• Catalytic part and built in RNA template
• attaches to end of chromosome
• DNA nucleotides complementary to the RNA template
  are added to the 3’ end of the lagging DNA strand
• Once the 3’ end is long enough, DNA polymerase adds the complementary nucleotides

Question 12

what does telomerase contain and how does it work

Answer 12

• Catalytic part and built in RNA template
• attaches to end of chromosome
• DNA nucleotides complementary to the RNA template
  are added to the 3’ end of the lagging DNA strand
• Once the 3’ end is long enough, DNA polymerase adds the complementary nucleotides

Question 12

what does telomerase contain and how does it work

Answer 12

• Catalytic part and built in RNA template
• attaches to end of chromosome
• DNA nucleotides complementary to the RNA template
  are added to the 3’ end of the lagging DNA strand
• Once the 3’ end is long enough, DNA polymerase adds the complementary nucleotides

Question 13

when does telomeres operate

Answer 13

Foetal development – rapid cell division
* Cancer – immortal cells
* Cellular aging
* (Not in adult cells)

Question 14

what are the 3 RNA polymerases in eukaryotic cells

Answer 14

RNA polymerase I – rRNA (5.8S, 18S and 28S)
2. RNA polymerase II – protein-coding genes, snoRNA, miRNA, siRNA and most snRNA
3. RNA polymerase III – tRNA, rRNA (5S), some snRNA

Question 15

what happens in initation of transcription of genes

Answer 15

RNA polymerase binds to the DNA of the gene at a promoter
* The promoter comes before (and slightly overlaps with) the transcribed region whose
transcription it specifies. It contains recognition sites for RNA polymerase or transcription
factors (TFs) to bind to
* The DNA opens up in the promoter so that RNA polymerase can begin transcription
* Once the transcription bubble has formed, the polymerase can start transcribing

Question 16

what is TATA box

Answer 16

The TATA box occurs between -30 and -25 elements of many eukaryotic promoters
o It is recognised by a general TF, allowing others and RNA polymerase II to bind
o The high frequency of A and T makes it easy to separate DNA strands in this region

Question 17

What are transcription factors

Answer 17

Basal/general TFs are proteins that are essential for transcription initiation. They bind to the promoter,
helping RNA polymerase to bind. Many promoters have a TATA box.

Question 18

How is gene expression regulated

Answer 18

Chromatin remodelling

• Enhance and silencer element
• methylation modification

Question 19

What does chromatin remodelling involve

Answer 19

Rearrangement of chromatin from condensed state to transceiptionall assessable state
Allow TF to bind and DNA binding proteins to access DNA and control gene expression

Question 20

What can alter structure of chromatic

Answer 20

Epigenetic modifications to histone proteins such as methylation,demethylation, acteylation and deacetylation can alter chromatin
- transcriptional activation/ repression

Epigenetics is covalent modification to DNA which impacts gene expression without affecting the underlying genetic sequence

Question 21

What do enhancer and silencer elements do

Answer 21

Regulate transcription through binding of multitude of TFs that activate or repress transcription

SILENCERS are antagonist of enhancers and repress transcription of genes when bound to TFs

Question 22

What is methylation

Answer 22

Occurs at Cytosine based of eukaryotic DNA

Common epigenetic signal that cells use to lock genes in the “off” position

Question 23

When does elongation occur

Answer 23

Once RNA polymerase is in position at the promoter

Walks along template strand 3’ to 5’

For each nucleotide, RNA adds a complementary RNA nucleotide to 3’ end of RNA strand, synthesising pre-mRNA in the 5’ to 3’ direction

Question 24

When does RNA Polymerase stop transcription

Answer 24

Once polymerase transcribed a sequence of DNA knwo bas terminato

In eukaryotes- RNA polymerase II takes place 1000 - 2000 nucleotides beyond the end of the gene being trnascrbed
This pre-mRNA tail is subsequently removed by cleavage during mRNA processing

RNA polymerase I and III need termination signals
Pol1 - 18 nucleotide sequence recognised by a termination protein
PolIII - mRNA hairpin similar to rho-independant termination of transcription in prokaryotes

Question 25

What happens after termination of prokaryotes

Answer 25

MRNA transcript are ready to be translated right away
5’-3’

Question 26

What happens to eukaryotes when transcriptions finishes

Answer 26

Translation happens in Christa
But RNA need to go through

• addition of 5’ cap
  ( protect RNA from degradation by ribonucleases and play a role in transport of mRNA out of cell) (cap-7’ methylguanosine and is added to the 5’ end of mRNA by 5’5’ bonding
• addition of polyA tail
  -AAUAAA (located near 3’ end of pre-mRNA)
  Adenylic acid molecules are added as a risk after enzyme action - polyadebylation

Question 27

What occurs in splicing

Answer 27

Remove introns from pre mRNA to become mRNA

Spliceosomes are involved in the linkage of 2 distinct exons after excision of introns

Bring together 2 breakpoints in pre-mRNA by binding to them
5’ splice site is cleaved: a phosphate on the 5’ terminus of intron binds to 2’ hydroxy group of a bear the 3’ terminus
A hydroxy group on the 3’ terminus of exon attacks the 3’ splice site

Alternatively - splicing mechanism which increases number of proteins encoded by the genome
(Amelogenin production)
tRNA and mRNA undergo post-transcriptional modification