6 - The mechanism of protein synthesis

Question 1

specifying amino acids

Answer 1

• mRNA cannot act as a physical template for amino acids

* tRNA is required to link mRNA and amino acids

Question 2

tRNA structure

Answer 2

• ~80 nucleotides in length
• Single stranded but base pairs form within the chain G-C, A-U etc.
• Clover leaf structure further folds to make L-shaped molecule
• Anticodon is at one end - base-pairs with codon
• Amino acid attachment site is the 3’ hydroxyl group at the end of the RNA chain

Question 3

tRNA specificity

Answer 3

• Each tRNA is specific for a single amino acid determined by its anticodon
• Specific attachment carried out by amino-acyl tRNA synthetases (activating enzymes)

Question 4

amino acyl tRNA synthetase

Answer 4

• One for each of the 20 amino acids 
• Binding sites for: 
1. specific tRNA(s), 
2. corresponding amino acid 
3. ATP • 2-step attachment process

Question 5

what is the 2- step attachment process?

Answer 5

1. ATP hydrolysed and amino acid joined to AMP

2. Correct tRNA binds and amino acid transferred from AMP to the tRNA

Question 6

wobble base pairing

Answer 6

Only occurs at third codon position between tRNA and mRNA.

G-U wobble pairing can occur

Question 7

ribosome

Answer 7

• Composed of rRNA and proteins
• 2 subunits – large and small
• Binds mRNA and amino acyl-tRNAs
• Catalyses stepwise formation of peptide bonds
• Moves in 5’-3’ direction along mRNA
• recognises the correct start codon, ensuring correct reading frame is used

Question 8

3 stages of protein synthesis

Answer 8

1. Initiation - small subunit binds mRNA and initiator amino acyl-tRNA then large subunit binds
2. Elongation - peptide bonds are formed as the ribosome moves along the mRNA
3. Termination - 1 of the 3 stop codons enters A-site and the completed protein is released

Question 9

initiation

Answer 9

1. Small ribosomal subunit binds mRNA near it’s 5’ end.
2. Initiator tRNA binds to AUG start codon
3. Large subunit binds so that the initiator tRNA fits into the P-site on the large subunit
   • Requires energy from GTP hydrolysis and initiation factors

Question 10

what is the role of initiation factors

Answer 10

help stabilise initiator tRNA and to assemble ribosome

Question 11

elongation

Answer 11

• Incoming aminoacyl tRNA base pairs with codon in the A-site - requires hydrolysis of GTP
• Peptide bond formed between amino group of the new amino acid and the COOH group of the amino acid in the P-site – catalysed by peptidyl transferase
• Growing polypeptide chain now in the A-site
• Translocation - tRNA in the P-site is ejected and the ribosome moves along the mRNA by precisely 1 codon – requires hydrolysis of GTP
• Growing chain now in the P-site and the A-site is free to accept the next incoming aminoacyl tRNA.

Question 12

Peptide bond formation

Answer 12

• Peptide bond formation catalysed by peptidyl transferase – an RNA enzyme (ribozyme)
• Proteins grow from amino (N) terminal to carboxy (C) terminal

Question 13

Termination

Answer 13

• Stop codon in A-site
• No tRNAs for stop codons
• Release factor enters A-site instead of amino acyl tRNA
• Water added to end of polypeptide chain
• Completed polypeptide released from tRNA in P-site
• Ribosome dissociates, 2 X GTP hydrolysed

Question 14

polyribosome (polysome)

Answer 14

A group of several ribosomes attached to, and translating, the same messenger RNA molecule.

Question 15

protein synthesis in eukaryotes

Answer 15

• Nuclear membrane – mRNAs transported to cytoplasm before translation occurs
• Several different organelles – proteins must be trafficked to correct site

Question 16

protein synthesis in prokaryotes

Answer 16

• No nuclear membrane – transcription and translation coupled
• No organelles – proteins diffuse through cytoplasm

Question 17

Targeting proteins to the ER

Answer 17

1. Polypeptide synthesis begins on a free ribosome in the cytosol.
2. An SRP binds to signal peptide, halting synthesis momentarily
3. The SRP binds to a receptor protein in ER membrane. This receptor is part of a protein complex that has a membrane pore and a signal-cleaving enzyme
4. The SRP leaves, and polypeptide resumes growing, meanwhile translocating across the membrane. (The signal peptide stays attached to the membrane.)
5. the signal-cleaving enzyme cuts off the signal peptide.
6. The rest of the completed polypeptide leaves the ribosome and folds into its final conformation.