Eukaryotic Transcription And Translation

Question 1

Compare a mitochondrial genome to a chloroplast genome

Answer 1

1. Mitochondria and chloroplasts divide independently of cell division
2. Numbers of mitochondria between 50-millions compared to 0-60 chloroplasts
3. Maternal inheritance in mitochondria
4. Chloroplast has a much larger genome

Question 2

How did organelles come to have DNA

Answer 2

1. Simple prokaryotes (proteobacterium) were endocytosed into a larger anaerobic cell
2. Proteobacterium + anaerobic cell have a symbiotic relationship due to the ability of the prokaryote to produce energy via aerobic respiration
3. Cyanobacteria had the ability to photosynthesise and respire

Question 3

Describe the types, structure and function of histones

Answer 3

1. Five types, H1, H2A, H2B, H3 and H4 histones
2. 4 pairs/octamer of histone proteins plus H1 histone that form the nucleosome
3. Wrap DNA around them, condensing them and making them inaccessible, but capable of unwrapping when DNA is required

Question 4

What is chromatin

Answer 4

1. The combination of DNA and the proteins it is associated with

Question 5

Give an example of non-histone protein function

Answer 5

1. Metaphase chromosome maintains its shape thanks to non-histone protein scaffold

Question 6

What is the visible manifestation when chromatin is loosened

Answer 6

1. Puff regions show uncoiled DNA and correspond to the high gene activity

Question 7

Contrast heterochromatin and euchromatin

Answer 7

1. Heterochromatin is strongly associated with histones and highly condensed, whereas euchromatin is uncoiled and less condensed

2 heterochromatin is therefore inaccessible and genetically inactive, whereas euchromatin is accessible and can be transcribed

3. Heterochromatin found in eukaryotes, whereas euchromatin found in both prokaryotes and eukaryotes

Question 8

Describe and explain the effects of histone modification (one tail per histone = 8 tails per nucleosome)

Answer 8

1. Lysine in histone tails can be acetylated and methylated
   -Acetylation results in heterochromatin shifting to euchromatin
   -Methylation results in tighter histone association and thus silences gene expression
2. Serine in histone tails can be phosphorylated
   -phosphatation prepares the chromatin for mitosis/meiosis (extreme condensation?)
3. Ubiquitination marks damaged or old proteins for destruction and/or recycling

Question 9

Explain the role of the chromatin remodelling complex in transcription

Answer 9

1. Chromatin remodelling complex uses ATP to bind to DNA, repositioning nucleosomes so that a transcription factor binding site is revealed
2. This initiates the assembly of the transcriptional apparatus

Question 10

Why is eukaryotic gene expression so variable

Answer 10

1. Complex organisms must respond to a wide range of changing stimuli
2. Multicellular organisms have cells that fulfill different purposes and need different proteins
3. Upregulation, downregulation and silencing are thus essential for multicellularity

Question 11

Give two examples of eukaryotic genes switching on and off

Answer 11

1. The photo activation of photosynthetic genes
2. Hormone-receptor interaction leads to a receptor signal that interacts with regulatory region of a hormone-responsive gene

Question 12

Differences in transcription between eukaryotes and prokaryotes

Answer 12

1. Transcription spatially separated from translation by the nuclear envelope
2. Different RNA polymerases are used in eukaryotes
   RNA pol II most active in eukaryotic transcription
3. Splicing, addition of 5’ cap and poly-A-tail occurs in eukaryotes (transcript processing)
4. More components in the transcriptional assembly in eukaryotes
5. Heterochromatin must be converted to euchromatin in order for transcription to occur
6. There is post-transcriptional control -

Question 13

Describe the three RNA pol forms

Answer 13

1. RNA pol I in the nucleolus and synthesises rRNA
   -not sensitive to a-amanitin
2. RNA pol II in the nucleoplasm and synthesises hnRNA (protein coding genes)
   -high sensitivity to a-Amanitin toxicity
3. RNA pol III in the nucleoplasm and synthesises tRNA and small RNAs (snRNAs)
   -medium sensitivity to a-amanitin

Question 14

Purpose of alternative splicing

Answer 14

To produce a variety of different mature mRNAs from the same pre-mRNA sequence

If certain splice factors are present, exons may be spliced out or spliced constitutively (kept), different 3’ or 5’ splice sites, introns kept

Question 15

What components make up the Pre-initiation complex

Answer 15

1. General transcription factors (TFIID/B/F/G/H) and RNA polymerase II bind to the TÂTA box, a region called the core promoter region of the gene.

Question 16

Functions of the general transcription factors GTFs

Answer 16

1. Position RNA poll II at the TATA box
2. Allow TATA-binding protein to then bend and separate DNA strands to initiate transcription
3. The equivalent of the sigma factors in prokaryotes

Question 17

Which TFs are utilised as a response to a stress stimulus that can also achieve co-ordinate control

Answer 17

1. Specific transcription factors bind to cis element sequences in the regulatory promoter region
2. This usually activates general transcription factors to bind to the core promoter (TATA box) initiating transcription
3. A specific transcription factor can bind to all cis sequences present independent on how close genes are to each other, thus one TF can regulate the expression of multiple genes at once.
4. Triggered usually due to cell draught, stress or light etc.

Question 18

How are transcription factors’ transcription regulated

Answer 18

1. Upon stresses or stimuli, the genes for the transcription factors can regulate the transcription OR translation of the TF gene
2. Usually the stability of the protein is stabilised by the draught/cold OR binding is facilitated more easily which allows them to fulfill their usual activity

Question 19

Sugggest a reason why organisms with similar genomes can have such similar phenotypes

Answer 19

1. Different patterns of TF expression and therefore levels of gene expression is the main reason phenotype between humans and chimps are so different despite sharing 99% genome similarity.

Question 20

What are all the components required for transvription

Answer 20

1. TFs
2. Poll II
3. GTFs
4. Co-activator - identified to be the ‘Mediator co-activator complex’

Question 21

Structure + Function of the Mediator

Answer 21

1. Brings TFs and RNA pol II together to more efficiently form the pre-initiation complex
2. Mediator is a multi-protein complex composed of a head attached to RNA poll II, middle and tail which interacts with transcription factors, together with a regulatory kinase attached.

Question 22

Give an example of a mutation in the mediator complex which lead to deleterious impact

Answer 22

1. Anti-freeze gene was not as highly expressed as the wild-type
2. Defective mediator tail subunit
3. Little effective communication between the TFs and the RNA Pol II
4. The antifreeze protein is plants was therefore lower and caused the plant respond negatively in the presence of cold

Question 23

Give an example of de-acetylation in flowers

Answer 23

1. Acetyl groups keep the FLC gene open for transcription
2. Transcribed and translated into a regulatory protein which represses flowering
3. Plants have a FLD gene which encodes a deacetylase enzyme which removes acetyl groups meaning FLC is not expressed, facilitating flowering

Question 24

Describe the structure+formation+general function of small RNAs

Answer 24

1. Double-stranded non-coding RNA
2. Formed via the DICER enzyme which chops a long dsRNA into pieces
3. General function is to silence/reduce gene expression at the post-transcriptional level

Question 25

Explain the formation and function of Small interfering RNAs (siRNA)

Answer 25

1. Formed via the cleavage of dsRNA by DICER
2. An siRNA combines with proteins to form RISC (RNA induced silencing complex)
3. Since siRNA is complementary to its own genes, this gene is targeted by RISC
4. RISC cleaves mRNA leading to degradation
5. SLICER catalyses cleavage triggered by siRNAs
6. Can ALSO attach to complementary mRNA sequence inducing histone methylation inhibiting transcription

Question 26

Explain the formation+function of micro RNAs (miRNAs)

Answer 26

1. Formed via the cleavage of single stranded RNA hairpins by DICER —> miRNAs
2. MiRNAs + proteins = RISC
3. The binding to mRNA is not perfect so translation is inhibited, but mRNA not cleaved

Question 27

Which mechanism leads to miRNAs degrading mRNA (miRNAs usually inhibit translation)

Answer 27

1. Some miRNAs complementary to an AU-rich region of the 3’ UTR end of mRNA activate DICER and can lead to mRNA degradation

Question 28

Other than gene inhibition what other roles does miRNAs fullfil

Answer 28

1. MiRNAs secreted via exocytosis outside the cell
2. Mediate cell-cell communication
3. De-regulation of miRNAs linked to cancer

Question 29

Describe the composition of the 70s and 80s ribosomes and explain what 70s or 80s mean

Answer 29

1. 70s is found in bacteria and has smaller subunits made of fewer rRNAs and proteins than 80s found under eukaryotes
2. 70s stands for the distance that the ribosomes sink when centrifuged in sucrose. Bigger = move more so 80s sinks more than 70s

Question 30

Describe the structure and role of tRNA

Answer 30

TRNA provides the complementary amino acid to the mRNA codon

Each amino acid has a specific tRNA

Amino acid bonds to the acceptor arm
The variable loop and tRNA arms help to differentiate between different tRNAs

Question 31

Describe the structure and roles of aminoacyl tRNA synthetases

Answer 31

1. Aminoacyl tRNA synthetase joins amino acid -AMP to the tRNA
2. There is a specific aminoacyl synthetase for each amino acid
3. AMP attachment required for enzyme to work
4. enzyme can identify incorrect amino acids and move them to the editing site where a-acid is hydrolysed and removed

Question 32

Explain the process of translation - Initiation

Answer 32

1. First codon on mRNA always AUG, methionine
2. Special initiator tRNA for start methionine that is different for other methionines
3. Bacteria one called N-formylmethionine
4. Small subunit binds to ribosome binding site with IF-3 to stop large subunit binding
5. Special initiator tRNA binds
6. Large subunit added
7. Subunit addition catalysed by initiation factors
8. Prokaryotes have a shine-dalgarno sequence which rRNA is complementary to helping ribosome bind to the mRNA
9. In eukaryotes the 5’ cap is recognised by IFs, polyA tail used for small subunit binding, and the Kozak sequence at the start codon is identified by ribosome

Question 33

Explain the process of elongation and translocation

Answer 33

1. Aminoacyl-tRNA bonds to the P site
2. New aminoacyl-tRNA bonds to A site
3. EF-1A hydrolyses GTP to GDP which gives energy to make peptide bond
   -Peptidyl transferase is the enzyme that catalyses peptide formation
4. EF-1B removes GDP and adds GTP back onto EF-1A
5. Deacylated tRNA falls off due to EF-2A GTP hydrolysis during translocation

Question 34

Explain the process of termination

Answer 34

1. RF-1 and RF-2 (releasing factor) occupies the A site
2. Amino acid in P site gains water instead of a peptide bond
3. RRF takes apart the subunits

Question 35

Describe the structure of+function of rRNA

Answer 35

1. RRNA catalyses peptide bond synthesis
2. Located on the small and large subunits
3. RRNA is folded such that G-U pairings can occur (weird folding)

Question 36

How does codon degeneracy physically happen

Answer 36

1. The third position in the anticodon undergoes relaxed binding
2. This is due to the curved shape of the anticodon that allows the third position to be the ‘wrong base’
3. Called wobble-pairing
4. Hence why most amino acids will corresponding sequences with the first two letters the same but the last letter different

Question 37

Explain the mechanism that deals with problematic mRNAs stuck in the ribosome in prokaryotes

Answer 37

1. Ribosome stalls when there is no stop codon
2. TmRNA is charged with Ala binding to the A site
3. Translocation can continue adding new amino acids ~10 alanines
4. TmRNA contains a stop codon
5. RF-1 and RF-3 together with GTP hydrolysis leads to termination
6. The alanine peptide is recognised and labelled for degradation

Question 38

What are the reasons for why the genetic code is in triplets

How was the triplet code proven

Answer 38

1. With 20 amino acids a doublet based code, only 16 combinations of sequences, and 3 would give 64 combos
2. By adding one nucleotide, there was a frame shift but removing one put the sequence back into frame

Adding two nucleotides muck it up

Adding three was very similar to wild type

Question 39

Explain nirenberg’s first experiment to examine which rna specified an amino acid

Answer 39

1. Polynucleotide phosphorylase used to make a single base polymer
2. Created a cell-free extract containing ribosomes and tRNA and an enzyme to destroy any other dna
3. One sample had radiolabelled amino acid
4. The radiolabelled protein would be made from a polypeptide made from only one amino acid
5. Made random copolymers = took a while with lots of uncertainty
6. Another scientist, Khorana, provided exact nucleotide sequences which could then be tested with each amino acid
7. tRNAs were used - code of mRNA was known, one a-acid radiolabelled so anticodon could be figured out and corresponded to the a-acid

Question 40

Describe the order of events during transcript processing

Answer 40

1. Intron excision and exon ligation
   - splicing carried out by the spliceosome
2. 5’ Cap added and poly A tail added by polyA polymerase