mRNAs

Question 1

Mechanism for creating mature mRNA?

Answer 1

Mature mRNA is created through splicing out the non- coding introns present.

GU and AG sequences represent the beginning and end of intron

1) U1 attaches to GU sequence in 5’ splice site which recruits U2AF
2) U2AF binds to AG sequence in 3’ splice site and recruits U2snRNP and SF1 which bind to precursor RNAs branch point site.
3) U4, U5 and U6 are recruited - spliceosome complex
4) U5 binds to both ends of exons
5) U6 displaces U1 and base pairs with U2 forming a lariat.

6) Cleavage happens through:
OH at the branch site attacks 5’ intron border
3’OH of exon 1 attacks 3’ intron-exon border

7) transesterification fuses exon sequences together. Intron is released

Question 2

Mechanisms in processing mRNA?

Answer 2

Before mRNA is ready for translation by ribosomes, it needs to go through processing via:

• addition of a methylated cap (protects mRNA from degradation)
• addition of a poly-adenylated tail

Poly-adenylation:

• Increases stability of mRNA, shorter tail means less stable
• Length shortens with each round of translation, so cell knows when it’s old and thus degrades it

Steps,

1) CPSF (cleavage and poly-adenylation specificity factor) and CsTF ( cleavage stimulatory factor) are bound to ‘poly A signal’ sequence and GU-rich downstream sequence on precursor RNA.
2) Cleavage factors 1 and 2 interact with CPSF, CsTF and precursor mRNA
3) recruitment of PAP (poly (A) polymerase ) which cleaves 3’ end and synthesises poly A tail through the addition of adenine residues
4) PABP2 (poly (A) binding protein) speeds up process

5) when tail reaches full length- signal informs PAP when to stop.
poly-adenylation process is complete

Question 3

The deal and stats surrounding JUNK protein?

Answer 3

• 98.8% of human genome is junk:
  Non coding … e.g non coding RNA
• Only 1% is coding
• composed of cis-acting sequences
• amount of junk increases with biological complexity

Question 4

What factors are involved in protein translation… and steps?

Answer 4

Codons:

• 64 possible combinations
• Start codon (AUG) and stop codons
• Codes for 20 amino acids

Ribosomes: 
- Composed of structural RNA and proteins 
- 60s complex: 28s rRNA, ~49 proteins 
3 cavities - exit, peptidyl, amino acyl 
- 40s complex : 18s rRNA, ~33 proteins 

tRNA: 
Transfer RNA 
- Amino acid 
- D loop, T loop 
- Anticodon loop: which contains 3 codons complementary to mRNA 

Steps,

1) tRNA and 40s complex bind to cap of mRNA and proceeds down searching for AUG (start) codon
2) 60s complex joins once first AUG codon is found. Binds so that tRNA is under Peptidyl cavity
3) second tRNA arrives with amino acids and binds to 60s ‘amino acyl’ cavity… a peptidyl bond is formed
4) 60s complex slides down: initial tRNA is now in exit cavity and can exit while the next tRNA is recruited.

5) cycle continues until stop codon is reached.
- a release factor breaks bond between the rRNA and amino acid sequence: ribosome falls apart

Question 5

What is a polyribosome?

Answer 5

A complex consisting of mRNA being simultaneously translated by multiple ribosomes

• resulting in the production of more proteins

Question 6

Mechanisms in place to ensure mRNA quality control?

Answer 6

1) mRNA turnover:
- Poly A tail becomes shorter with each round of translation.
- Deadenylation stimulates activity of a 3’-5’ exonuclease
- This results in decapping which recruits 5’-3’ exonuclease
- mRNA components are recycled

2) Nonsense mediated decay:
- premature stop codon: results in a trunkated protein which can be detrimental.
- splicing event removes exon function junction (EFJ) from mRNA
- This sequence is still within mRNAs with premature stop codons, thus it gets identified by ribosome and ejected
- leads to decapping of mRNA recruits 5’-3’ exonuclease