Synthesis of Proteins

Protein Synthesis

Learning Outcomes

• Explain how primary, secondary, tertiary, and quaternary structures of proteins influence their form and function.
• Model translation, the process by which a cell reads a mature mRNA sequence to form a polypeptide.
• Describe the involvement of cellular organelles in the production of a functional protein.
• Describe the impact of different types of mutation on protein structure and gene function.
• Illustrate how manipulation of DNA can be used for research and biotechnological applications.

Protein Translation and Mutation

• Labeling elements of a ribosome during one of the stages of translation.
• Using a codon table to work out amino acid sequences of genes.

Activity: Protein Translation and Mutation

• Part 1 instructions: Label a ribosome from one of the stages of translation and then decide where to place the appropriate tRNAs.
• Part 2 instructions: Use a codon table to work out the amino acid sequences for wild type and mutated genes, then state the type of mutation that has affected the gene and whether that mutation is likely to have an impact on gene function.

Part 1: Translation

• Label the figure with the suggestions provided; not all labels are needed as some elements will not be found on the figure.

• Determine which of the tRNAs in the table should go in positions 1, 2, and 3 of this ribosome, decide whether they are staying in, entering, or exiting the ribosome at this stage.

• Positions on the ribosome:

  • Position 1: Exit (E) site
  • Position 2: Peptidyl (P) site
  • Position 3: Amino acyl (A) site

Poll Questions on tRNA placement:

• Q1. Which of the eight tRNA molecules should go in the amino acyl (A) site (position 3 in the table)?
• Q2. Which of the eight tRNA molecules should go in the peptidyl (P) site (position 2 in the table)?
• Q3. Which of the eight tRNA molecules should go in the exit (E) site (position 1 in the table)?

Part 2: Translation and Mutation

• Use the codon table to work out amino acid sequences.
• Create an amino acid sequence for a tiny, complete gene.
• You will be given mutant versions of that gene. Fill in the amino acid sequences, highlight each triplet codon in the mRNA, the change in the mRNA code, and any differences in the amino acid code.
• Determine the type of mutation that occurred and whether or not you think the mutation will affect gene function.

Wild Type Gene

• 5’ G C A U G A C C G C G A A A U A A G C 3’
• Q4. What is the 3-letter abbreviation of the amino acid that always starts a polypeptide?
• Q5. What is the mRNA codon for this first amino acid?
• NB: This is not the entire mRNA sequence; there would be UTRs at both ends e.g. 5’ uaucuaugcgugacGCAUGACCGCGAAAUAAGCugcaugcguaugcuguagcgaacugg 3’ (5'UTR … ATG … TAG … 3'UTR)
• The translation START codon is in the gene’s first exon (exon 1).

Wild Type Amino Acid Sequence

• Q6. What is the amino acid sequence of this mini gene?
• Correct Answer: MET (M) – THR (T) – ALA (A) – LYS (K) – STOP
• 5’ G C A U G A C C G C G A A A U A A G C 3’

Mutation 1

• 5’ G C A U G A C C G C G A U A U A A G C 3’
• Amino acid sequence: MET (M) – THR (T) – ALA (A) – ILE (I) – STOP
• Q7. What type of mutation is mutation 1?
• Answer: Nonsense, causing a premature stop codon.
• Would this mutation affect the gene’s function? Yes.

Mutation 2

• 5’ G C A U G G C G A A A U A A G C 3’
• Amino acid sequence: MET (M) – ALA (A) – LYS (K) – STOP
• Mutation 2 is an in-frame deletion – one amino acid is removed but the gene’s reading frame is retained
• Any deletion or insertion that is a multiple of three (3, 6, 9 etc) will NOT affect the gene’s reading frame

Mutation 3

• 5’ G C A U G A C G G C G A A A U A A G C 3’
• Amino acid sequence: MET (M) – THR (T) – ALA (A) – LYS (K) – STOP
• Mutation 3 is a Silent mutation (also known as a Synonymous mutation) and will NOT affect the gene’s function

Mutation 4

• 5’ G C A U G A C C U G C G A A A U A A G 3’
• Amino acid sequence: MET (M) – THR (T) – CYS (C) – GLU (E) – ILE (I)
• Q8. What type of mutation is seen in mutation 4?
• Answer: 1 bp insertion leading to a frameshift
• Where will this protein end? It continues until it reaches a stop codon.

Mutation 5

• 5’ G C A U G A C C G C G U A A U A A G C 3’
• Amino acid sequence: MET (M) – THR (T) – ALA (A) - STOP STOP
• Mutation 5 is a single base pair substitution leading to a NONSENSE mutation, where an amino acid is replaced by a STOP codon, called a PREMATURE STOP codon.
• Nonsense mutations lead to TRUNCATED proteins.

Coding mutations in Wilson Disease

• Wilson disease shows considerable allelic heterogeneity

• Most Wilson disease patients are compound heterozygotes e.g. ATP7BH1069Q / ATP7BR778L (Chang and Hahn, Handb Clin Neurol. 2017;142:19-34).

• They are homozygous for ATP7B mutations but have two different mutations

• Of the most common Wilson disease mutations there are:

  • 5 nonsense mutations
  • 3 frameshift mutations
  • 4 indels
  • 34 missense mutations

• Q9. Why might missense mutations be more frequent than nonsense and frameshift mutations?

Coding mutations in Wilson Disease

• Q10a. Which exon is the biggest hotspot for pathogenic (disease-causing ) mutations?

• Q10b. Why might this exon be more prone to pathogenic mutations? (Chang and Hahn, Handb Clin Neurol. 2017;142:19-34.)

• Exon 8 contains the most pathogenic mutations.

• It encodes two important transmembrane domains

• It is also one of the biggest exons!!

• The frequency of pathogenic mutations is actually higher in exon 13 (Chang and Hahn, Handb Clin Neurol. 2017;142:19-34).

Coding mutations in Wilson Disease

• The most common Wilson disease mutation ATP7BH1069Q causes the protein to be misfolded and retained in the endoplasmic reticulum, inhibiting its Golgi and plasma membrane functions.
• Specific kinase inhibitors can restore proper ATP7B cellular localization and could therefore be used to treat Wilson disease. (Chesi et al., Hepatology. 2016 Jun;63(6):1842-59.)

Recombinant DNA technology in Wilson disease diagnosis

• (Bao-yu Peng et al. Acta Pharmacol Sin 2017; 39: 117–12.)

Recombinant DNA technology in Wilson disease treatment

• (Goswami Front. Oncol., 24 April 2019 | https://doi.org/10.3389/)