Protein Translation and Post-Translational Modifications

Question 1

The genetic code

Answer 1

• Links nucleic acid and protein info
• 3 nucleotides (codons) encode an amino acid
• Read from 5’ to 3’ end of mRNA
• Degenerate
• Some codons are synonymous
• Codon bias (some organisms prefer a specific codon for a.a)
• Start = AUG (coding strand = ATG)
• Stop = UAA, UAG, UGA

Question 2

tRNA Structure

Answer 2

Transfer, adaptor molecules that recognize codons are mRNA

• 73-93 nucleotides
• L-shaped 3D structure
• Phosphorylated 5’ end
• 3’ CCA arm (a.a attachment site)
• Anti-codon loop (base pairs with a codon)

Question 3

Wobble Base-Pairing

Answer 3

• Some tRNAS recognize more than one codon
• Usually in 3rd position of codon
• Allows for pt mutations not as serious because there is no change in the a.a formed

Question 4

tRNA Charging

Answer 4

Formation of a peptide bond b/w free a.a is thermodynamically unfavourable
- Activated intermeds for protein synthesis are a.a esters called aminoacyl tRNA (charged tRNA)

Question 5

Aminoacyl tRNA synthetase

Answer 5

• A.As are first activated by adenylation
• Aminoacyl-AMP is transferred to tRNA
• All this is catalyzed by Aminoacyl tRNA synthetase

Question 6

tRNA Charging Steps

Answer 6

1. Activation by adenylation
2. Transfer to tRNA
   - Highly exergonic rxn (spon)

Overall rxn: a.a + ATP + tRNA + H2O < > aminoacyl-tRNA + AMP + 2Pi

Question 7

How do aminoacyl-tRNA synthetases confer specificity?

Answer 7

“Double Sieve” = has 2 sites for checking if Thr is added

• Specific interactions b/w side chain OH of Thr and active site will prevent binding of Val
• Ser is small enough to fit in editing site where it will be removed, Thr doesn’t fit = not removed

Question 8

The Ribosome

Answer 8

Euk have: 80S with 40S and 60S subunits
Prok have: 70S with 30S and 50S subunits
rRNA plays a catalytic role

Question 9

Protein Synthesis: Initiation

Answer 9

• Shine-Dalgano site before START codon
• Bacterial mRNA is polycistronic (encodes for >1 protein)
• Begins with N-formylmethionine (fMet)
• fMet is removed as polypeptide grows
• Euks begin with normal Met
• IFs (initiation factors) + fMet-tRNAf + mRNA + 30S form initiation complex
• 50S subunit joins
• GTP is hydrolyzed

Question 10

Protein Synthesis: Elongation

Answer 10

EF-Tu-GTP binds to aminoacyl-tRNAs and delivers them to A site

• Protects tRNA from cleavage and ensures correct bping at A site
• Hydrolysis of GTP releases the aminoacyl-tRNA

N IS NUCLEOPHILE IN A SITE!!!!

Question 11

Protein Synthesis: Termination

Answer 11

• RF1 or RF2 (release factors) recognize the stop codons, bind at the A-site, and stim the hydrolysis of the final ester linkage b/w peptide and last tRNA = peptide chain cleaved
• RF3 is a GTPase catalyzes removal of the RFs and completed protein
• After release, the protein folds and may be post-translationally modified

Question 12

Post-Translational Protein Modifications

Answer 12

• Side chains that are most commonly modified include: R-OH, R-NH2, or R-SH
• Influences chemical properties, results in changes of net charge, conformation, binding and/or function
• Most are reversible and catalyzed by a specific enzyme