DNA & Genomics II

Question 1

Genetic Code

Answer 1

• Gene is a specific nucleotide sequence on DNA that codes for a specific AA sequence
• Central Dogma: DNA gene - codon in mRNA sequence - primary AA structure - unique protein conformation - protein function
  Characteristics:
  1. Triplet code
  2. Universal
  3. Degenerate (same AA coded by more than 1 codon)
  4. Non-overlapping (read as successive codons)
  5. Continuous
  6. Start codon AUG; Stop codons UAG/UGA/UAA

Question 2

Transcription Definition

Answer 2

• Process in which nucleotide sequence of DNA is used as a template to direct synthesis of single-stranded RNA
• Synthesis in 5’ —> 3’ catalyse by RNA polymerase
  Gene includes:
• Promoter as recognition site
• Termination Sequence that stops RNA synthesis
• Transcription Unit

Question 3

Transcription Process

Answer 3

Initiation
1. RNA polymerase attaches to promoter
2. RNA polymerase unzips and separates 2 strands of DNA double helix by breaking H bonds between CBPs
Elongation
3. One DNA strand used as template strand for synthesis of complementary mRNA strand
4. Free nucleotides matched up with DNA template via CBP + mention A=U, T=A, C=G
5. RNA polymerase catalyses formation of phosphodiester bonds between adjacent ribonucleotides to form sugar-phosphate backbone
6. New mRNA strand synthesised in 5’ —> 3’ direction with DNA reannealing after transcription

Termination
7. After RNA polymerase transcribed through termination sequence, mRNA chain is released and RNA pol dissociates

Question 4

Translation

Answer 4

Initiation
1. Amino acid activation: A specific AA is covalently bonded to its specific tRNA at its 3’ CCA stem with specific anticodon to form an aminoacyl tRNA. This attachment is catalyses by a specific aminoacyl tRNA synthetase, achieving double specificity
2. Small ribosomal subunit and initiator tRNA carrying methionine bind to newly synthesised mRNA strand
3. Anticodon UAC of initiator tRNA CBPs with start codon AUG
4. Binding of large ribosomal subunit forms a translation initiation complex
5. Initiator tRNA positioned at peptidyl P site, leaving aminoacyl A site vacant for next aminoacyl tRNA
Elongation
6. 2nd tRNA with a specific anticodon binds to mRNA codon via CBP at A site
7. A peptide bond is formed between Met and second AA catalyses by peptidyl transferase on large subunit. Met dissociates from initiator tRNA
8. Ribosome translocates by 1 codon in 5’ —> 3’ direction
- 1st tRNA shifted to E site and released into cytosol
- 2nd tRNA shifts to P site and carried growing polypeptide chain
- Incoming aminoacyl-tRNA carrying next AA can now enter empty A site
Termination
9. Process continues until stop codon UAA/UAG/UGA exposed at A site. Specific release factors enter A site, causing hydrolysis of bond between polypeptide chain and tRNA in P site
10. Polypeptide released from ribosome to complete its folding into necessary secondary and tertiary structure. Ribosome disassembles into subunits
11. Polyribosomes can form from cluster of ribosomes simultaneously translating mRNA for a faster rate of protein synthesis

Question 5

Gene Mutations

Answer 5

• Change in sequence of DNA nucleotide bases, mRNA codons and AA primary sequence. This affects protein folding in tertiary structure, changing 3D conformation and function of protein
  A. Single Base Substitution
  B. Addition and Deletion which can result in frameshift mutations that can cause all meaning to be lost/truncated polypeptide to form
  C. Inversion

Question 6

Sickle Cell Anaemia

Answer 6

• Single base substitution in B-globin Gene where thymine replaced by adenine (CTC —> CAC on template DNA) results in 6th codon on mRNA to GAG to GUG
• Change from hydrophilic glutamate to hydrophobic valine, affects tertiary structure and the bonds formed between R groups
• Change in conformation results in HbS
• Normal circular biconcave shape of RBC becomes sickle shaped under low [O2].
• When RBC moves to low [O2], hydrophobic patches stick together, causing HbS to polymerase into abnormal rigid rod-like fibres

Question 7

Effects of Sickle Cell Anaemia

Answer 7

1. Sickle RBCs are more fragile and are actively destroyed in spleen, resulting in shorter lifespan. This leads to a shortage of RBCs and poor O2 transport, causing patient to suffer from anaemia and lack of energy
2. Pointed sickle shaped RBCs often get lodged in capillaries, causing blockages that can lead to organ damage