Mechanism of protein synthesis

• How to specify amino acids?

  mRNA cannot act as physical template for amino acids so adapters are require to link mRNA and amino acids. Adapters are transfer RNAs (tRNAs)
• What is the struture of tRNA?

  ~80 nucleotides in length

  single stranded but base pair form within the chain G-C, A-U

  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
• how is tRNA specific?

  each tRNA is specific for a single amino acid determined by its anticodon
• How does a tRNA become attached to the correct amino acid?

  specific attachment carried out by amino-acyl tRNA synthetase (activating enzyme)
• how is an amino acid attached to tRNA?

  amino acyl-tRNA synthetases
• What are the binding sites on amino acid?

  specific tRNA, corresponding amino acid, ATP
• How are amino acids and tRNA attached?

  1. ATP hydrolysed amino acids joined to AMP
  2. correct tRNA binds and amino acid transferred from AMP to the tRNA
• how many tRNAs are there?

  40-45
• what is wobble base pairing?

  The third nucleotide (3' end)of a codon in mRNA and the first position anticodon of tRNA can exhibit some flexibility in base pairing
• what allows wobble base pairing?

  The redundancy of the genetic code
• base in wobble position G pairs with?

  U or C in thrid base in codon
• where is the wobble position?

  first base on the anticodon (5' end)
• if C is in the wobble position what is in the third base in the codon?

  G
• if a is in the wobble position what is in the third base in the codon?

  U
• if U is in the wobble position what is in the third base in the codon?

  A or G
• if I is in the wobble position what is in the third base in the codon?

  U, C or A
• What is the structure of the ribosome?

  Composed of rRNA and proteins

  contains 2 subunits (large and small)

  binds mRNA and amino acyl-tRNAs

  catalyses stepwise formation of peptide bonds (amino acids added from N terminus to C terminus)

  moves in 5' to 3'
• how does a ribosome ensure a correct reading frame?

  the correct start codon
• structure of ribosome

  A) P site (peptidyl-tRNA binding site)

  B) E site (exist site)

  C) mRNA binding site

  D) smaller subunit

  E) large subunit

  F) A site (aminoacyl-tRNA binding site)

  G) exit tunnel

  
• what is the exit tunnel for on the ribosome?

  growing polypeptide
• what are the 3 stages of protein synthesis?

  initiation, elongation, termination
• describe initiation

  small subunit binds mRNA and initiator amin acyl-tRNA the larger subunit binds
• Describe elongation

  Peptide bond are formed as the ribosome moves along the mRNA
• describe termination

  one of the three stop codons enters A-site and the completed protein is released
• stages of initiation

  1. small ribosomal subunit binds mRNA near its 5' end (recognises sequence signal just 5' of AUG start codon)
  2. initiator tRNA binds to AUG start codon
  3. large subunit binds so that the initiatior tRNA firs into the P-site on the large subunit

  • requires energy from GTP hydrolysis and proteins called initiation factors (help stabilise initiator tRNA and to assemble ribosome)
• stages of elongation

  1. incoming aminoacyl tRNA base pairs with codon in the A-site - requires hydrolysis of GTP
  2. 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
  3. growing polypeptide chain is now in the A-site
  4. Translocation - tRNA in the P-site is ejected and the ribosome moves along the mRNA by precisely one codon - requires hydrolysis of GTP
  5. growing chain is now in the P-site and the A-site is free to accepty the next aminoacyl tRNA
• how is a peptide bond formed?

  catalysed by peptidyl transferase

  formed between COOH in Peptidyl tRNA and Amino acyl tRNA

  protein grown from amino (N) terminal to carboxy (C) terminal
• describe termination

  Stop codon is in A site, there are no tRNA for a stop codon, release factor enter A-site (instead of an amino acyl tRNA)

  water added to end of polypeptide chain

  completed polypeptide released from tRNA in P-site

  Ribosome dissociates, 2 X GTP hydrolysed
• what are polyribosomes?

  Clusters of ribosomes translating the same mRNA simultaneously.
• How does proteins synthesis in prokaryotes and eukaryotes?

  Eukaryotes

  • nuclear membrane - mRNAs transported to cytoplasms before translation occurs
  • several different organelles - proteins must be trafficked to correct site

  prokaryotes

  • no nuclear membrane- transcription and translation are coupled
  • no organelles - proteins diffuse through cytoplasm
• Protein secretion via the endoplasmic reticulum

  1. Polypeptide synthesis begins on a free ribosome in the cytosol

  2. A signal recognition particle binds to signal, halting synthesis momentarily

  3. The SRP binds to a receptor protein the ER membrane. This receptor is a part of a protein complex that has membrane pore and 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 complete polypeptide leaves the ribosome and fold into its final conformation
• elongation cycle

  A) codon recognition

  B) 2 GTP

  C) 2 GDP

  D) peptide bond formation

  E) GTP

  F) GDP

  G) translocation

  H) ribosome ready for next amino acyl tRNA

  I) amino end of polypetide

  
• what is translocation?

  translocation refers to the movement of the ribosome along the mRNA molecule during the elongation phase of protein synthesis