Protein Degradation
What is a Degron?
Bonus points:
How does it tie into the N-end rule
A degron is a specific sequence of AA that indicates a protein should be broken down.
Bonus points:
It ties into the N-end rule as the composition of the N-terminal amino acid is a very significant contributor to half-life
In broad strokes how does a mRNA vaccine work?
By understanding the composition of the major target of our immune system. You can determine what mRNA makes that target, deliver that mRNA to the cell, and have a ribosome produce it. The body targets the foreign object and builds defense against it so when you encounter the actual disease, you are protected.
What is the difference between siRNA and miRNA
natural, originates from the cell
Less specific, one miRNA targets multiple mRNAs
Imperfectly complimentary
Represses translation
Originates from single-stranded hairpins precursors
Originating from non-coding regions
siRNA:
Synthetic, artificially implanted
Highly specific, often targeting only one mRNA
Perfectly complementary
Cleaves mRNA
Originates from a double-stranded structure
Both:
20-25 nt
Prepared by Dicer enzyme
What are the 2 ways a 5' cap can be added to mRNA vaccines
1) Vaccina capping enzyme
2) In-vitro co-transcriptional cap addition
-Via competing first base addition of cap analog: Clean cap AG reagent
What is the importance of k48 vs k63 ubiquitin chains?
Bonus points:
How many Ubs must be linked to be recognized for protein degradation?
K48: Breakdown (me fr)
K63: Signaling
Bp: 4
How do you make a mRNA for mRNA vaccines
1) Make the plasmid
2) Cut out the promoter and gene with restriction enzymes or perform PCT
3) Treat with T7 polymerase to make mRNA
4) do necessary post-translational modifications
Explain the process of attaching a Ubiquitin to the target protein.
Bonus points:
What residue of ubiquitin is attached to what residue on the target protein?
1) ATP is added to the C-terminus of Ub, activating it and releasing ppi
2) The E1 protein adds replaces the AMP attaching to the Ub
3) E2 replaces E1
4) E3 ligase transfers Ub to target proteins' lysine
Bonus points:
Carboxyl terminal Glycine of Ub to Lysine of targer
Explain the key components and aspects of the 19s and 20s subunits of the proteosome.
19s:
Contains the Ub receptors, binds Ub chains
Contains an Isopeptidase (DUB) that removes the Ub
Uses ATP to unfold protein and direct it into the 20s
20s:
3 Active sites in Beta subunit, one for acidic AA, one for basic and one for Hydrophobic
Processive breakdown of proteins
Breaks down to 7-9 peptides before release