Subsection- Replication
Case Scenario 1:
A 10-year-old boy is brought to the clinic with complaints of growth retardation, sun sensitivity, and frequent skin blistering on sun exposure. On examination, he has hyperpigmented, dry skin with multiple lesions on sun-exposed areas. Laboratory studies reveal defective DNA repair mechanisms, and he is diagnosed with xeroderma pigmentosum (XP).
- a) What is DNA replication, and what are its key features?
b) Which enzymes and steps are involved in normal DNA replication?
c) How does a defect in DNA replication/repair explain the clinical findings in this patient?
Answer
- a) DNA replication & its key features:
- DNA replication is the process of synthesizing a new DNA strand using the existing strand as a template before cell division.
- Key features:
- Semi-conservative: each daughter DNA has one parental and one newly synthesized strand.
- Bidirectional: proceeds in both directions from the origin.
- Requires a primer (short RNA segment) to initiate synthesis.
- Occurs during the S-phase of the cell cycle.
- b) Enzymes & steps of DNA replication:
Major steps & enzymes:
| Step | Enzyme/Protein involved |
| Unwinding of DNA | Helicase breaks hydrogen bonds. |
| Prevention of strand reannealing | Single-strand binding proteins (SSBs) |
| Relief of supercoiling | Topoisomerase (gyrase) |
| Primer synthesis | Primase lays down RNA primers. |
| DNA synthesis (elongation) | DNA polymerase III (prokaryotes) — adds nucleotides in 5′→3′ direction. |
| Primer removal & gap filling | DNA polymerase I (prokaryotes) |
| Joining Okazaki fragments | DNA ligase forms phosphodiester bonds. |
| Proofreading | DNA polymerase (3′→5′ exonuclease activity). |
- c) Defect in DNA replication/repair in xeroderma pigmentosum:
- In XP, there is a defect in the nucleotide excision repair (NER) pathway, which normally removes UV-induced thymine dimers.
- Although the primary replication machinery is intact, damaged DNA cannot be properly repaired before or during replication → mutations accumulate.
- UV exposure causes DNA lesions that replication forks cannot bypass efficiently, leading to cell death or carcinogenesis.
- Clinical features:
- Sun sensitivity & skin blistering due to accumulation of DNA damage.
- Increased risk of skin cancers
Subsection: Transcription
Case Scenario 2:
A 45-year-old man working in a pesticide factory presents with complaints of fatigue, weight loss, and frequent infections. On examination, he has pallor and mild splenomegaly. Bone marrow biopsy shows suppression of RNA synthesis. History reveals chronic exposure to actinomycin D (a transcription inhibitor).
- a) What is transcription and what are its key features?
b) Which enzymes and steps are involved in transcription?
c) How do inhibitors of transcription (like actinomycin D) affect this process, and what is their clinical significance?
Answer
- a) Transcription & its key features:
- Transcription is the process of synthesizing RNA from a DNA template by RNA polymerase.
- Key features:
- Synthesizes RNA in the 5′ → 3′ direction.
- Complementary to the DNA template (antisense) strand.
- Does not require a primer.
- In prokaryotes: occurs in the cytoplasm; in eukaryotes: in the nucleus.
- In eukaryotes, different RNA polymerases synthesize different types of RNA.
- b) Enzymes & steps of transcription:
Steps of transcription:
| Step | Description & Enzymes |
| Initiation | RNA polymerase binds to promoter region (with sigma factor in prokaryotes). |
| Elongation | RNA polymerase moves along DNA, adding ribonucleotides complementary to template strand. |
| Termination | In prokaryotes, either Rho-dependent or Rho-independent termination; in eukaryotes, specific signals terminate transcription. |
Eukaryotic RNA polymerases:
| Polymerase | RNA synthesized |
| RNA Pol I | rRNA |
| RNA Pol II | mRNA, some snRNA |
| RNA Pol III | tRNA, 5S rRNA |
- c) Effect of transcription inhibitors & clinical significance:
- Actinomycin D (Dactinomycin):
- Binds to DNA at the transcription initiation complex and inhibits elongation by RNA polymerase.
- Blocks both prokaryotic and eukaryotic RNA synthesis.
- Used clinically as an anticancer drug because it inhibits rapidly dividing tumor cells.
- Other inhibitors:
- Rifampicin: inhibits prokaryotic RNA polymerase.
- α-Amanitin: inhibits eukaryotic RNA polymerase II → toxic (mushroom poisoning).
Case Scenario 3:
Sarah, a 35-year-old woman, visits her physician with complaints of persistent cough, fever, fatigue, and weight loss for several weeks. She reveals that her father was recently diagnosed with tuberculosis (TB). The physician prescribes her rifampin as part of the treatment regimen.
Questions:
- a) Which step of genetic information processing is inhibited by rifampin?
b) Explain the process of transcription in eukaryotic cells.
c) How does transcription in eukaryotes differ from that in prokaryotes?
d) Write a short note on post-transcriptional modifications.
Answer
- a) Step inhibited by rifampin:
- Rifampin inhibits the process of transcription.
- It specifically binds to the β-subunit of prokaryotic RNA polymerase, blocking the initiation of RNA synthesis.
- Since TB is caused by Mycobacterium tuberculosis (a prokaryote), rifampin effectively halts its RNA production and thus protein synthesis.
- b) Transcription in eukaryotic cells:
- Transcription is the synthesis of RNA from a DNA template using RNA polymerase.
- Occurs in the nucleus in eukaryotes.
Steps:
- Initiation — RNA polymerase II binds to the promoter (with help of transcription factors) and unwinds DNA.
- Elongation — RNA polymerase moves along the template strand, synthesizing RNA in the 5′→3′ direction.
- Termination — RNA polymerase releases the newly synthesized pre-mRNA after reading termination signals.
- c) Differences between eukaryotic & prokaryotic transcription:
| Feature | Eukaryotes | Prokaryotes |
| Location | Nucleus | Cytoplasm |
| RNA polymerases | 3 types (I, II, III) | Single RNA polymerase |
| Initiation | Needs transcription factors | Direct binding to promoter |
| RNA processing | Pre-mRNA undergoes processing | mRNA is ready to translate |
| Coupling with translation | Separated from translation | Coupled with translation |
- d) Post-transcriptional modifications in eukaryotes:
After transcription, the primary transcript (pre-mRNA) undergoes processing to become mature mRNA:
5′ Capping: Addition of a 7-methylguanosine cap to the 5′ end — protects RNA & aids in ribosome binding.
Polyadenylation: Addition of a poly(A) tail at the 3′ end — stabilizes mRNA & aids export.
Splicing: Removal of introns (non-coding sequences) and joining of exons (coding sequences) by the spliceosome.
Subsection: Translation
Case Scenario 4:
Jenny, a 10-year-old girl, is brought to the emergency department with complaints of sore throat, fever, and difficulty breathing. On examination, a thick grayish membrane is seen covering her tonsils and pharynx. The healthcare provider suspects diphtheria, starts appropriate treatment, and explains that the bacterial toxin inhibits an important step of genetic information processing.
- a) Which step in the processing of genetic information is inhibited by the diphtheria toxin?
b) Describe the phases of protein synthesis.
c) Briefly describe other inhibitors of protein synthesis.
Answer
- a) Step inhibited by diphtheria toxin:
- Diphtheria toxin inhibits translation (protein synthesis) at the elongation phase.
- It catalyzes ADP-ribosylation of elongation factor-2 (EF-2), inactivating it → halting translocation of the ribosome on mRNA in eukaryotic cells.
- This prevents the addition of further amino acids to the growing polypeptide chain.
- b) Phases of protein synthesis: activation, initiation, elongation, and termination
- c) Inhibitors of protein synthesis:
| Inhibitor | Target & effect |
| Diphtheria toxin | Inhibits EF-2 (eukaryotes) — blocks elongation. |
| Chloramphenicol | Inhibits peptidyl transferase (prokaryotes). |
| Tetracycline | Blocks aminoacyl-tRNA binding to A-site (prokaryotes). |
| Streptomycin | Causes misreading of mRNA (prokaryotes). |
| Erythromycin | Blocks translocation (prokaryotes). |
| Puromycin | Causes premature chain termination (both prokaryotes & eukaryotes). |
| Cycloheximide | Inhibits peptidyl transferase (eukaryotes). |
Subsection: Mutation
Case Scenario 5:
A 6-year-old boy is brought to the hospital with complaints of progressive muscle weakness, difficulty standing up, and delayed motor milestones. He is diagnosed with Duchenne Muscular Dystrophy (DMD), caused by a mutation that produces a premature stop codon in the dystrophin gene, leading to defective protein synthesis.
- a) What is translation, and what are its key features?
b) Describe the steps of translation in eukaryotic cells and the major molecules involved.
c) How do defects in translation contribute to diseases like Duchenne Muscular Dystrophy?
Answer
- a) What is translation?
- Translation is the process of protein synthesis, where the information encoded in mRNA is decoded to produce a specific polypeptide chain (protein) on ribosomes.
- Key features:
- Occurs in the cytoplasm.
- Requires mRNA, ribosomes, aminoacyl-tRNAs, and energy (ATP & GTP).
- Synthesizes polypeptides from the N-terminal to C-terminal end, reading mRNA from 5′ → 3′.
- b) Steps of translation in eukaryotes:
Initiation:
- The small ribosomal subunit binds to the 5′ cap of mRNA with the help of initiation factors.
- The initiator tRNA carrying methionine (Met-tRNA) binds to the start codon (AUG).
- The large ribosomal subunit joins to form the complete 80S ribosome.
Elongation:
- Aminoacyl-tRNAs bring amino acids to the ribosome according to the codons.
- Peptidyl transferase (rRNA of large subunit) forms peptide bonds between amino acids.
- Ribosome moves along mRNA (translocation), and the polypeptide chain elongates.
Termination:
- When a stop codon (UAA, UAG, UGA) is encountered, release factors bind.
- The completed polypeptide is released, and the ribosome dissociates.
- c) Defects in translation & DMD:
- In DMD, a nonsense mutation introduces a premature stop codon in the dystrophin mRNA.
- This leads to premature termination of translation → production of a truncated, nonfunctional dystrophin protein.
- The lack of functional dystrophin weakens muscle cell membranes, leading to muscle degeneration.
Case Scenario 6:
A 35-year-old man with known sickle cell trait is admitted to the hospital with complaints of severe joint pain, fatigue, and breathlessness. He is diagnosed with a sickle cell crisis.
- a) What is the defect in the globin chain of hemoglobin in this condition?
b) What is a point mutation? Explain the types of point mutations with examples.
c) How does the specific point mutation in sickle cell disease lead to its clinical consequences?
Answer
- a) Defect in the globin chain:
- In sickle cell disease/trait, there is a point mutation in the β-globin gene of hemoglobin (HBB gene).
- Specifically, at codon 6 of β-globin, GAG → GTG, leading to substitution of glutamic acid by valine.
- This altered hemoglobin is called HbS (sickle hemoglobin).
- b) Point mutation & its types:
Point mutation — a change in a single nucleotide of DNA.
Types:
| Type of point mutation | Definition | Example |
| Silent | Change in codon without change in amino acid | AAA → AAG (both = Lys) |
| Missense | Change in codon → different amino acid | GAG → GTG (glutamic acid → valine) in sickle cell disease |
| Nonsense | Change in codon → stop codon → truncated protein | β-thalassemia |
- c) How the mutation causes disease:
- The missense mutation in β-globin causes HbS, which tends to polymerize under low oxygen conditions, distorting red blood cells into a sickle shape.
- Consequences of sickled RBCs:
- Decreased deformability → blockage of small vessels → pain (vaso-occlusive crisis).
- Shortened RBC lifespan → hemolytic anemia.
- Increased risk of infarctions, infections, and organ damage.