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FIRST YEAR MBBS BIOCHEMISTRY - BFUHS UNSOLVED PAPERS

Complete Answers

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SECTION 1: RNA/DNA STRUCTURE (from Page 125)

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Q11. Name the different arms of tRNA and mention their functions. (2007)

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Q12 & Q13. Synthetic Purine Analogues and their Clinical Significance / Purine Analogues (2007, 2005)

1. 6-Mercaptopurine (6-MP)
   • Analogue of hypoxanthine
   • Mechanism: inhibits purine de novo synthesis (blocks IMP conversion to AMP and GMP); incorporated into DNA
   • Clinical use: leukemia (ALL), Crohn's disease
   • Activated by HGPRT enzyme
2. Azathioprine
   • Prodrug converted to 6-MP in the body
   • Clinical use: immunosuppression in organ transplants, rheumatoid arthritis, IBD
3. 6-Thioguanine
   • Analogue of guanine
   • Clinical use: acute leukemia
4. Allopurinol
   • Analogue of hypoxanthine
   • Inhibits xanthine oxidase (prevents uric acid formation)
   • Clinical use: gout, hyperuricemia
5. Acyclovir
   • Analogue of guanosine
   • Inhibits viral DNA polymerase (selective for herpes virus thymidine kinase)
   • Clinical use: herpes simplex, varicella zoster
6. Vidarabine (Ara-A)
   • Adenine arabinoside; inhibits viral DNA polymerase

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Q14. Structure of DNA (2005)

1. Double helix: Two antiparallel polynucleotide strands coiled around a common axis
2. Backbone: Sugar-phosphate backbone on the outside (deoxyribose + phosphodiester bonds)
3. Bases: Inside, perpendicular to helix axis; stacked 0.34 nm apart
4. Base pairing (Chargaff's rules):
   • A = T (2 hydrogen bonds)
   • G ≡ C (3 hydrogen bonds)
5. Pitch: 3.4 nm per complete turn; 10 base pairs per turn
6. Grooves: Major groove (wide, ~22 Å) and minor groove (narrow, ~12 Å); protein binding sites
7. Diameter: ~2 nm
8. Antiparallel orientation: One strand 5'→3', the other 3'→5'

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SECTION 2: GENETICS - DNA REPLICATION, RECOMBINATION AND REPAIR

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Q1. Base Excision Repair (BER) (2023)

1. Recognition: DNA glycosylase recognizes and cleaves the damaged base, creating an AP (apurinic/apyrimidinic) site
2. AP site cleavage: AP endonuclease cleaves the phosphodiester bond 5' to the AP site
3. End processing: DNA deoxyribose phosphodiesterase (dRPase) removes the sugar-phosphate remnant
4. Gap filling: DNA polymerase beta fills the gap using the intact strand as template
5. Ligation: DNA ligase seals the nick

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Q2. Salient Features of the Genetic Code (2021)

1. Triplet code: Each codon consists of 3 nucleotides (64 possible codons)
2. Degenerate/Redundant: Multiple codons specify the same amino acid (e.g., leucine has 6 codons). Only Met (AUG) and Trp (UGG) have single codons
3. Non-overlapping: Codons are read sequentially without overlap
4. Non-punctuated: No punctuation marks between codons; read continuously
5. Universal: Same code used by almost all organisms (exceptions: mitochondria, some protozoa)
6. Unambiguous: Each codon specifies only one amino acid
7. Initiator codon: AUG (methionine) - also serves as start codon
8. Terminator codons: UAA, UAG, UGA (stop codons - no corresponding tRNA)
9. Wobble hypothesis (Crick): The 3rd position of the codon and 1st position of the anticodon can wobble, allowing one tRNA to recognize multiple codons
10. Commaless: Reading frame is set from the start and has no internal stops

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Q3. Why do Silent Mutations have No Effect on Phenotype? (2021)

1. Degeneracy of the genetic code: Multiple codons encode the same amino acid. A mutation in the 3rd codon position (wobble position) often still codes for the same amino acid (e.g., GCU, GCC, GCA, GCG all code for Alanine)
2. Protein structure unchanged: Since the amino acid sequence is identical, the protein folds the same way and retains full function
3. No change in function: Enzyme activity, receptor binding, structural properties remain unaltered
4. Example: CGA → CGG (both code for Arginine) - this change is silent

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Q4. Missense Mutations (2019, 2004)

• Conservative: Similar amino acid substituted (e.g., Val → Ile; both nonpolar) - minimal effect
• Non-conservative: Chemically different amino acid substituted - often significant effect

• Can alter protein folding, stability, active site, or function
• May cause loss of function, gain of function, or dominant negative effects

1. Sickle Cell Anemia: Glu → Val at position 6 of beta-globin (GAG → GTG). This single change causes HbS polymerization under deoxygenation
2. PKU: Mutations in phenylalanine hydroxylase gene
3. Osteogenesis Imperfecta: Gly → another amino acid in collagen

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Q5. Frameshift Mutation (2018, 2009)

• Insertion of 1-2 nucleotides shifts the reading frame +1 or +2
• Deletion of 1-2 nucleotides shifts the frame -1 or -2
• All codons downstream of the mutation are read incorrectly

• Completely different amino acid sequence downstream of the mutation
• Premature stop codon is likely encountered, truncating the protein
• Usually results in a non-functional protein

• Duchenne Muscular Dystrophy (DMD): frameshift in the dystrophin gene
• Tay-Sachs disease: 4-bp insertion in HEXA gene

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Q6. Point Mutations (2017, 2010)

1. Transitions: Purine ↔ Purine (A↔G) or Pyrimidine ↔ Pyrimidine (C↔T) - more common
2. Transversions: Purine ↔ Pyrimidine - less common but more disruptive

1. Silent (synonymous): No amino acid change (due to degeneracy)
2. Missense: Different amino acid substituted
3. Nonsense: Creates a premature stop codon (UAA, UAG, UGA) → truncated protein

• Spontaneous deamination (C→U), depurination
• Mutagens: alkylating agents, base analogues (5-BU), UV radiation, X-rays
• Replication errors

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Q7. Cell Cycle (2016)

• G1 phase (Gap 1): Cell growth; synthesis of proteins needed for DNA replication; restriction point (R point) - decision to commit to division
• S phase (Synthesis): DNA replication occurs; histone synthesis; centrosome duplication; duration ~6-8 hours
• G2 phase (Gap 2): Further growth; synthesis of mitotic apparatus proteins; repair of replication errors; ~4-5 hours
• G0 phase: Quiescent, non-dividing state

• Prophase, Metaphase, Anaphase, Telophase → Cytokinesis

1. G1/S checkpoint: Checks DNA integrity before replication (p53, Rb proteins)
2. G2/M checkpoint: Checks DNA replication completeness
3. Spindle assembly checkpoint (SAC): All chromosomes attached to spindle

• Cyclins (regulatory subunit) + CDKs (cyclin-dependent kinases, catalytic subunit)
• CDK4/6 - Cyclin D: G1 progression
• CDK2 - Cyclin E: G1/S transition
• CDK1 - Cyclin B: M phase (MPF - maturation promoting factor)

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Q8. DNA Polymerases (2015)

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Q9. Nucleosome Assembly (2015)

• ~147 bp of DNA wound 1.65 turns around a histone octamer
• Histone octamer: 2 copies each of H2A, H2B, H3, H4 (core histones)
• Linker DNA (~20-80 bp) connects adjacent nucleosomes
• Histone H1 binds at the entry/exit point of DNA

1. H3-H4 tetramer first: Two H3-H4 dimers assemble on DNA first, forming a tetrasome
2. H2A-H2B dimer addition: Two H2A-H2B dimers join on either side
3. Complete nucleosome core particle formed (~10 nm "beads on a string")
4. Chromatin assembly factors (CAF-1) assist in vivo, especially during replication
5. Histone chaperones (nucleoplasmin for H2A-H2B; CAF-1 for H3-H4) prevent non-specific DNA binding

• 2 nm DNA → 11 nm nucleosome fiber → 30 nm solenoid (H1-stabilized) → 700 nm condensed chromosome

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Q10. Effects of Mutations (2012)

1. Silent/Synonymous: No amino acid change; neutral
2. Missense: Different amino acid; variable effect
3. Nonsense: Premature stop codon; truncated protein
4. Frameshift: Altered reading frame; non-functional protein
5. Splice site mutations: Altered mRNA processing; exon skipping

1. Loss-of-function: Enzyme/protein activity reduced/absent (usually recessive) - e.g., PKU, albinism
2. Gain-of-function: Protein becomes constitutively active (usually dominant) - e.g., certain cancers, achondroplasia
3. Dominant-negative: Mutant protein interferes with normal protein function

1. Lethal: Embryonic/fetal death
2. Deleterious: Disease (sickle cell, cystic fibrosis)
3. Neutral: No noticeable effect
4. Beneficial: Evolutionary advantage (very rare)

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Q11. DNA Replication in Prokaryotes - Process and Inhibitors (2011)

1. Initiation: Begins at oriC (origin of replication); DnaA protein recognizes and melts the origin
2. Unwinding: Helicase (DnaB) unwinds the double helix; creates two replication forks moving bidirectionally
3. Stabilization: SSB (single-strand binding proteins) stabilize single-stranded DNA; Topoisomerase I/II relieves positive supercoils ahead of the fork
4. Primer synthesis: Primase synthesizes a short RNA primer (5-10 nt)
5. Elongation:
   • DNA Pol III synthesizes both strands 5'→3'
   • Leading strand: Synthesized continuously toward the fork
   • Lagging strand: Synthesized discontinuously as Okazaki fragments (~1000-2000 nt in prokaryotes)
6. Primer removal: DNA Pol I removes RNA primers (5'→3' exonuclease); fills gaps
7. Ligation: DNA Ligase seals nicks (uses NAD+ in bacteria)
8. Termination: Ter sequences and Tus protein halt replication; replication ends when forks meet

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Q12. Xeroderma Pigmentosum (XP) (2009)

1. Damage recognition (XPA, XPC)
2. Helicase unwinds around lesion (XPB, XPD - part of TFIIH)
3. Dual incision 5' and 3' of lesion
4. Gap filling (Pol δ/ε) and ligation

• Skin: Extreme photosensitivity; sunburn with minimal UV; freckling, skin atrophy, telangiectasia
• Eyes: Photophobia, conjunctivitis, corneal clouding
• Neurological (some types): Progressive neurological degeneration
• Malignancy: 1000-fold increased risk of skin cancers (SCC, BCC, melanoma); median age ~8 years

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Q13. Eukaryotic Primary Transcript (hnRNA/pre-mRNA) (2008)

• Directly transcribed from DNA by RNA Pol II
• Much larger than mature mRNA
• Unstable; processed in the nucleus before export

1. 5' Capping: 7-methylguanosine (m7G) cap added to the 5' end
   • Function: Protection from exonucleases; ribosome recognition for translation initiation; mRNA export
2. 3' Polyadenylation: A poly-A tail (100-250 adenylate residues) added to the 3' end after cleavage ~20 nt downstream of AAUAAA signal
   • Function: mRNA stability; nuclear export; translation efficiency
3. Splicing (removal of introns): Pre-mRNA splicing removes intervening sequences (introns) and joins exons
   • Carried out by the spliceosome (snRNPs: U1, U2, U4, U5, U6)
   • Two-step transesterification reactions
4. RNA editing: Post-transcriptional modification of nucleotides (e.g., C→U in apoB mRNA)

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Q14. Differences Between Prokaryotic and Eukaryotic Replication (Tabular) (2007)

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Q15. Eukaryotic DNA Replication (2006)

1. Multiple origins (replicons): Thousands of origins fire simultaneously or in waves, allowing the large genome to be replicated in hours
2. Licensing factor control: Origins fire only once per S phase; MCM complex (helicase) is loaded in G1 (origin licensing); fired and removed in S phase to prevent re-replication
3. Chromatin remodeling: Nucleosomes must be disassembled ahead of the fork and reassembled behind; histone chaperones (CAF-1) assist
4. Telomere replication: Telomerase (reverse transcriptase with RNA template) extends chromosome ends to prevent shortening
5. Cell cycle control: Replication is tightly linked to CDK activity (CDK2-Cyclin E initiates S phase)
6. PCNA: Sliding clamp that processivity factor for Pol δ

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SECTION 3: TRANSCRIPTION AND TRANSLATION

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Q1. Alternative Splicing (2023)

1. Exon skipping: One or more exons are excluded from the mature mRNA (most common)
2. Alternative 5' splice site: Different 5' splice site used
3. Alternative 3' splice site: Different 3' splice site used
4. Intron retention: An intron is retained in the mRNA
5. Mutually exclusive exons: Only one of two exons is included

• SR proteins (exonic splicing enhancers): Promote exon inclusion
• hnRNP proteins (exonic splicing silencers): Promote exon exclusion
• Tissue-specific and developmental stage-specific

• ~95% of human multi-exon genes undergo alternative splicing
• Dramatically expands the proteome from ~20,000 genes
• Examples: Troponin T (cardiac vs skeletal), DSCAM (Drosophila, ~38,000 isoforms), Bcl-x (pro-apoptotic Bcl-xS vs anti-apoptotic Bcl-xL), Calcitonin/CGRP

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Q2. Okazaki Fragments (2023)

• Prokaryotes: 1000-2000 nucleotides
• Eukaryotes: 100-200 nucleotides
• Each begins with a short RNA primer (~10 nt)

1. Primer removal: RNase H and FEN1 (eukaryotes) or DNA Pol I (prokaryotes) remove RNA primers
2. Gap filling: DNA Pol fills the gap with DNA
3. Ligation: DNA ligase joins adjacent fragments via phosphodiester bond (uses ATP in eukaryotes, NAD+ in prokaryotes)

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Q3. Regulation of Transcription and Post-Transcriptional Modifications in Eukaryotes (2022)

A. Regulation of Transcription

1. Chromatin remodeling: Histone acetylation (HATs) opens chromatin; histone deacetylation (HDACs) condenses it. DNA methylation at CpG islands silences genes.
2. General transcription factors (GTFs): TFIID (recognizes TATA box via TBP), TFIIA, TFIIB, TFIIF, TFIIE, TFIIH assemble at promoter to form preinitiation complex (PIC)
3. Promoter elements: TATA box (~25 bp upstream), initiator element, CAAT box (~75 bp upstream), GC box
4. Transcriptional activators and repressors: Bind enhancers/silencers (can be thousands of bp away); communicate via mediator complex with PIC
5. RNA Pol II CTD phosphorylation: Phosphorylation at Ser5 (initiation), Ser2 (elongation) coordinates transcription and RNA processing

B. Post-Transcriptional Modifications

1. 5' Capping (m7G cap): Added co-transcriptionally; protects mRNA, aids ribosome binding
2. 3' Polyadenylation: Poly-A polymerase adds 200-250 A residues; mRNA stability and export
3. Splicing: Spliceosome removes introns; alternative splicing generates diversity
4. RNA editing: Enzymatic modification (ADAR: A→I; APOBEC: C→U)
5. Nuclear export: mRNA exported via nuclear pore with export factors (TAP/NXF1)
6. mRNA degradation control: miRNAs target mRNAs for translational repression or degradation (RISC complex)

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Q4. Why is DNA Replication Semiconservative? (2021)

1. E. coli grown in heavy nitrogen medium (¹⁵N) for many generations → all DNA labeled with ¹⁵N (heavy)
2. Transferred to light nitrogen (¹⁴N) medium
3. DNA isolated after each generation and centrifuged in CsCl density gradient

• After 1 generation: Single intermediate-density band (¹⁵N-¹⁴N hybrid)
• After 2 generations: Equal amounts of intermediate and light bands
• This pattern fits only semiconservative replication

• Helicase unwinds and separates the two strands
• Each parental strand serves as a template for new synthesis
• New strand is synthesized complementary to the parental template
• Result: two identical double-stranded DNA molecules, each with one old + one new strand
• This ensures perfect fidelity of genetic information transfer

• Conservative (both parental strands stay together): Not observed
• Dispersive (parental DNA scattered randomly): Not observed

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Q5 & Q7. Post-Translational Modifications in Eukaryotes (2021, 2018, 2015)

• Insulin: Signal peptide removed; C-peptide cleaved from proinsulin
• Collagen: Pro-α chains → hydroxylation of Pro/Lys → glycosylation → triple helix → procollagen cleavage → tropocollagen
• Coagulation factors: Glu → γ-carboxyglutamate (Vit K dependent) for calcium binding

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Q6. RNA Polymerase (2019)

• Single type; core enzyme: α₂ββ'ω subunits
• Sigma (σ) factor added to form holoenzyme; recognizes promoter (-10 and -35 elements)
• Does NOT need a primer; can start new RNA chains de novo
• Inhibited by: rifampicin (binds β subunit, blocks chain initiation), actinomycin D

• Contains heptapeptide repeats (YSPTSPS)
• Phosphorylation coordinates transcription elongation with capping, splicing, and polyadenylation

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Q8. Footprinting (2017)

1. DNA fragment is end-labeled with radioactive ³²P at one end
2. Protein (transcription factor, repressor, etc.) is incubated with labeled DNA to allow binding
3. DNase I is added at low concentration - cleaves accessible (unprotected) phosphodiester bonds randomly
4. Protein is removed by denaturation; DNA is recovered
5. Samples are run on denaturing polyacrylamide gel
6. Gel is autoradiographed
7. "Footprint" = region of gel where bands are absent (protected from DNase cleavage by the bound protein)

• Identify promoter elements and transcription factor binding sites
• Map protein-DNA interactions genome-wide (DNase-seq)
• Study drug-DNA interactions

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