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FIRST YEAR MBBS BIOCHEMISTRY - COMPLETE ANSWERS

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SECTION A: TRANSCRIPTION & TRANSLATION

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9. Write a Short Note on Transcription Factors (2016)

1. General (Basal) Transcription Factors - Required for transcription of all genes. Examples: TFIIA, TFIIB, TFIID (contains TATA-binding protein/TBP), TFIIE, TFIIF, TFIIH. They assemble into a preinitiation complex at the promoter.
2. Specific Transcription Factors (Activators/Repressors) - Regulate specific genes. Examples: p53, NF-kB, Sp1.

• DNA-binding domain - recognizes specific DNA sequences (e.g., helix-turn-helix, zinc finger, leucine zipper motifs)
• Activation domain - interacts with the transcription machinery to activate or repress transcription
• Dimerization domain - allows protein-protein interaction

• RNA Pol II (responsible for mRNA synthesis) requires multiple general transcription factors.
• TFIID binds the TATA box (located ~25 bp upstream of start site).
• Other factors assemble sequentially, forming the preinitiation complex.
• TFIIH has helicase and kinase activities - it unwinds DNA and phosphorylates RNA Pol II to initiate transcription.

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10. Define/Discuss Splicing (2016, 2006)

1. The pre-mRNA contains exons and introns. The spliceosome (a complex of snRNPs - small nuclear ribonucleoproteins) recognizes splice sites.
2. A 2'-OH of an adenosine residue (branch point, ~30 nt upstream of 3' splice site) attacks the 5' splice site - creating a lariat intermediate.
3. The freed 3'-OH of the upstream exon attacks the 3' splice site, joining the two exons.
4. The lariat (intron) is released and degraded.

• 5' splice site: GU (donor site)
• 3' splice site: AG (acceptor site)
• Branch point: adenosine residue

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11. Discuss SNPs (Single Nucleotide Polymorphisms) (2016)

• Occur approximately once every 300 bases throughout the human genome
• More than 10 million SNPs exist in humans
• Must occur in at least 1% of the population to be called an SNP (otherwise it is a mutation)
• Can be synonymous (silent) or non-synonymous (missense/nonsense)

• Disease susceptibility: SNPs are associated with diseases like diabetes, cancer, hypertension
• Pharmacogenomics: SNPs in drug-metabolizing enzyme genes (e.g., CYP2D6) determine individual drug response and toxicity
• Forensic medicine: SNPs serve as genetic markers for identification
• GWAS (Genome-Wide Association Studies): Use SNPs to identify disease-associated genomic regions

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12. Discuss Eukaryotic Basal Transcription Complex Formation; Post-Translational Modifications (2015)

1. TFIID (containing TBP - TATA-binding protein) binds the TATA box at -25 to -30 from start site. TBP bends DNA ~90°.
2. TFIIA and TFIIB stabilize TBP binding.
3. RNA Pol II - TFIIF complex is recruited.
4. TFIIE and TFIIH join, completing the preinitiation complex (~2 million Da).
5. TFIIH (with helicase activity) unwinds DNA; its kinase phosphorylates the CTD (C-terminal domain) of RNA Pol II at Ser-5 to initiate transcription.
6. After promoter clearance, further phosphorylation at Ser-2 of CTD marks elongation phase.

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13. Describe Transcription in Bacteria vs. Mammalian Transcription (2012)

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14. Write a Short Note on Translation (short note)

• mRNA (template), ribosomes (site), tRNA (adapter), amino acids, ATP, GTP, initiation/elongation/termination factors

• In prokaryotes: 30S subunit binds Shine-Dalgarno sequence on mRNA; initiator tRNA (fMet-tRNA^fMet) binds AUG start codon; 50S joins.
• In eukaryotes: 43S preinitiation complex scans from 5' cap to find AUG; 60S subunit joins. Initiator amino acid is Met (not formylated).

• Aminoacyl-tRNA delivery (A site): EF-Tu (prokaryotes) or eEF-1α (eukaryotes) delivers aminoacyl-tRNA to A site. GTP hydrolysis provides energy.
• Peptidyl transfer: Peptidyltransferase activity of 23S/28S rRNA (ribozyme) forms peptide bond between P-site peptide and A-site amino acid.
• Translocation: EF-G (prokaryotes) or eEF-2 (eukaryotes) moves ribosome 3 nt in 3' direction. Peptidyl-tRNA moves from A to P site; empty tRNA leaves via E site.

• Stop codons (UAA, UAG, UGA) are recognized by release factors (RF1, RF2 in prokaryotes; eRF1 in eukaryotes).
• Peptidyl-tRNA bond is hydrolyzed, releasing the polypeptide.
• Ribosome dissociates.

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15. Write a Short Note on Chaperones (2011)

1. Hsp70 family (Heat Shock Protein 70 kDa):
   • Bind newly synthesized unfolded polypeptides
   • Prevent premature folding and aggregation
   • Require ATP hydrolysis to release the substrate
   • Examples: Hsp70, BiP (in ER), DnaK (E. coli)
2. Hsp60/Chaperonins:
   • Large barrel-shaped complexes (GroEL/GroES in bacteria; Hsp60/Hsp10 in mitochondria; CCT/TRiC in cytoplasm)
   • Provide an isolated chamber for a single protein to fold correctly
   • ATP-dependent
3. Hsp90:
   • Primarily involved in stabilizing and activating signaling proteins like steroid hormone receptors and kinases
4. Small Hsps: Hsp27 - oligomeric complexes that bind unfolded proteins and hold them for refolding by Hsp70.

• Facilitate correct folding of newly synthesized proteins
• Prevent aggregation during stress (heat, oxidative stress)
• Assist in translocation of proteins across membranes
• Help degrade misfolded proteins via ubiquitin-proteasome pathway

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16/17. Briefly Discuss / Features of Genetic Code (2008, 2010)

1. Triplet code (codon = 3 nucleotides): Each amino acid is specified by a set of 3 nucleotides (codon). With 4 bases, 4³ = 64 possible codons exist. 61 code for amino acids; 3 are stop codons.
2. Specificity (Unambiguous): Each codon codes for only ONE specific amino acid. There is no ambiguity.
3. Degeneracy (Redundancy): Most amino acids are coded by MORE than one codon (synonymous codons). Only Met (AUG) and Trp (UGG) have a single codon. This protects against point mutations.
4. Non-overlapping: Each nucleotide belongs to only one codon. Codons are read sequentially without overlap.
5. Commaless (No punctuation): There are no gaps or spacers between codons. Reading starts from a fixed AUG and continues uninterrupted.
6. Universality: The genetic code is essentially the same in all organisms - from bacteria to humans. Exceptions: mitochondria (UGA codes for Trp; AGA/AGG are stop codons).
7. Polarity: Read 5' → 3' on mRNA.
8. Start codon: AUG (codes for Met in eukaryotes; fMet in prokaryotes) - also defines the reading frame.
9. Stop codons (Nonsense codons): UAA, UAG, UGA - do not code for any amino acid; signal end of translation.
10. Wobble hypothesis (Crick): The third base of the codon and first base of the anticodon can form non-Watson-Crick base pairs, explaining why fewer than 61 tRNA species are needed.

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18. Define Exons (2007, 2006)

• Exons contain the coding sequences (CDS) as well as the 5' and 3' untranslated regions (UTRs).
• After transcription, the pre-mRNA is processed by splicing to remove introns, leaving only exons in the mature mRNA.
• In humans, the average gene has ~8 exons.
• Through alternative splicing, different combinations of exons can give rise to different protein isoforms from a single gene.

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19. Define Introns (2007, 2006)

• Introns begin with GU and end with AG (GT-AG rule at the DNA level).
• They contain the branch point sequence (YNYURAY, where A is the branch point adenosine).
• Introns make up ~24% of the human genome; exons only ~1.5%.
• Functions: regulate gene expression, alternative splicing, some introns are self-splicing (ribozymes - Group I and II introns).

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20/21. Describe/Write about Genetic Code (2005, 2004)

• 64 total codons
• AUG = start codon (Met)
• UAA, UAG, UGA = stop codons
• The remaining 61 codons specify the 20 standard amino acids

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SECTION B: REGULATION OF GENE EXPRESSION

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1. Write a Short Note on Probes (2023)

1. DNA probes - single-stranded DNA fragments
2. RNA probes (riboprobes) - more stable than DNA probes
3. Oligonucleotide probes - short synthetic 18-30 nucleotide sequences; highly specific

• Radioactive labels: ³²P (most common; detected by autoradiography)
• Non-radioactive labels: Biotin (detected by streptavidin-enzyme), Digoxigenin (DIG), Fluorescent dyes (used in FISH - Fluorescence In Situ Hybridization)

• Southern blotting - detect specific DNA sequences
• Northern blotting - detect specific mRNA sequences
• FISH (Fluorescence In Situ Hybridization) - detect chromosomal abnormalities
• Microarray/DNA chips - thousands of probes simultaneously for gene expression profiling
• PCR - as primers
• Prenatal diagnosis - detect mutations in genetic diseases

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2. Write a Short Note on Regulated Expression of Gene (2023)

• Promoters and enhancers control when, where, and how much RNA Pol binds
• Transcription factors activate or repress gene expression
• In prokaryotes: operon model (lac operon, trp operon)
• In eukaryotes: chromatin remodeling, histone acetylation/deacetylation, methylation

• Alternative splicing - multiple protein isoforms from one gene
• mRNA stability - AU-rich elements in 3' UTR destabilize mRNA (e.g., cytokine mRNAs)
• miRNA (micro RNA) and siRNA (small interfering RNA) - bind to complementary mRNA sequences and cause degradation or translational silencing (RNA interference/RNAi)

• Control of initiation factors (e.g., eIF2α phosphorylation during stress halts global translation)
• Iron response elements (IRE) in ferritin mRNA - when iron is low, IRP binds IRE and blocks ferritin translation

• Protein modifications (phosphorylation, ubiquitination) regulate protein activity and stability

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3 & 6. Explain Lac Operon (2017, 2015, 2009, 2005)

[Regulatory gene (lacI)] -- [Promoter (P)] -- [Operator (O)] -- [lacZ] -- [lacY] -- [lacA]

• lacZ - encodes β-galactosidase (cleaves lactose → glucose + galactose)
• lacY - encodes permease (transports lactose into cell)
• lacA - encodes transacetylase (function unclear)
• Operator: where repressor binds
• Promoter: where RNA Pol binds
• CAP site: upstream of promoter, for catabolite activator protein binding

• The lac repressor (product of lacI gene) is active.
• It binds to the operator and physically blocks RNA Pol from transcribing.
• The operon is OFF.

• Lactose is metabolized to allolactose (true inducer).
• Allolactose binds to the lac repressor, causing a conformational change.
• The inactive repressor can no longer bind the operator.
• RNA Pol transcribes the structural genes → produces polycistronic mRNA → three enzymes are made.
• The operon is ON.

• When glucose is absent: adenylyl cyclase is active → high cAMP levels.
• cAMP binds CAP (Catabolite Activator Protein / CRP - cAMP Receptor Protein).
• The cAMP-CAP complex binds the CAP site (upstream of promoter) and greatly stimulates RNA Pol binding.
• Operon is strongly ON.
• When glucose is present: cAMP levels are low → CAP is inactive → poor transcription even if lactose is present.
• This ensures glucose is used preferentially (glucose catabolite repression).

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4. Discuss Prokaryotic and Eukaryotic Gene Regulation (2016)

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5. Write a Short Note on Helix-Turn-Helix Motif (2015)

• Consists of two α-helices connected by a short turn (β-turn of ~4 amino acids)
• The C-terminal helix ("recognition helix") fits into the major groove of DNA and makes specific base contacts
• The N-terminal helix stabilizes the interaction by lying across the top of the DNA

• HTH proteins often function as dimers, with two recognition helices fitting into successive major grooves on the same face of the DNA (separated by ~3.4 nm)
• Specific amino acid residues on the recognition helix form hydrogen bonds with specific base pairs

• Zinc finger: Uses Zn²⁺ to stabilize a finger-like structure that inserts into DNA major groove (e.g., Sp1, TFIIIA)
• Leucine zipper (bZIP): Two α-helices dimerize via leucine-rich regions; basic region contacts DNA (e.g., c-Fos, c-Jun)
• Helix-loop-helix (HLH): Similar to HTH but with larger loop; common in developmental regulators (e.g., Myc, MyoD)

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SECTION C: RECOMBINANT DNA AND BIOTECHNOLOGY

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1. Describe the Procedure and Uses of Southern Blotting (2023)

1. DNA Extraction and Restriction Digestion: Genomic DNA is extracted and cut with restriction endonucleases into fragments.
2. Gel Electrophoresis: DNA fragments are separated by size using agarose gel electrophoresis (smaller fragments migrate further).
3. Denaturation: The gel is treated with NaOH to denature double-stranded DNA into single strands.
4. Transfer (Blotting): Single-stranded DNA is transferred from gel to a nitrocellulose or nylon membrane (by capillary action, vacuum, or electroblotting). This creates a faithful replica of the gel pattern on the membrane.
5. UV Crosslinking/Baking: DNA is fixed to the membrane.
6. Hybridization: The membrane is incubated with a labeled probe (complementary to the target sequence) under stringent conditions. The probe hybridizes to complementary sequences.
7. Washing: Non-specific probe is washed away.
8. Detection: If radioactive probe (³²P) is used - autoradiography. If non-radioactive (biotin/DIG) - enzyme-linked colorimetric/chemiluminescent detection.

• Diagnosis of genetic disorders (e.g., sickle cell anemia, thalassemia)
• RFLP analysis and DNA fingerprinting
• Detection of specific genes
• Gene mapping
• Detection of viral DNA in clinical samples
• Paternity testing

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2 & 4. Polymerase Chain Reaction (PCR) - Procedure and Uses (2023, 2018, 2011, 2022)

• Template DNA (containing the target sequence)
• Two oligonucleotide primers (forward and reverse, flanking the target, ~18-25 bp each)
• Thermostable DNA polymerase (Taq polymerase from Thermus aquaticus)
• All four dNTPs (dATP, dGTP, dCTP, dTTP)
• MgCl₂ (cofactor), buffer

• High temperature breaks hydrogen bonds between DNA strands
• Double-stranded DNA separates into two single strands

• Temperature is lowered to allow primers to bind (hybridize) to their complementary sequences on the template
• Forward primer binds one strand; reverse primer binds the other strand, flanking the target

• Taq polymerase extends from 3' end of each primer, synthesizing new DNA strands
• 72°C is the optimal temperature for Taq polymerase

• 20 cycles = ~10⁶ copies
• 30 cycles = ~10⁹ copies
• Each cycle takes ~5-10 minutes; a 30-cycle PCR is complete in 2-3 hours

1. Diagnosis of infectious diseases: HIV, TB, COVID-19, Hepatitis, Malaria
2. Prenatal diagnosis: Detect genetic disorders (Down syndrome, cystic fibrosis) from fetal DNA
3. Cancer diagnosis: Detect oncogenes, translocations (e.g., BCR-ABL in CML)
4. Forensic medicine: DNA fingerprinting from trace evidence (blood, hair)
5. Paternity testing
6. Blood bank screening: Detect viruses in donated blood
7. HLA typing for organ transplantation

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3 & 6. DNA Fingerprinting and Why Used for Paternity Testing (2022, 2019)

• Certain regions of the human genome contain short repetitive DNA sequences (2-6 bp repeats) called STRs/microsatellites.
• The number of repeats at each locus varies between individuals.
• These polymorphic repeat regions are inherited from parents in a Mendelian fashion.

1. Extract DNA from sample (blood, saliva, semen, hair root, buccal swab)
2. Cut DNA with restriction enzymes → different-sized fragments depending on number of repeats
3. Electrophoresis → separation by size
4. Southern blotting → transfer to membrane
5. Hybridize with labeled probe complementary to the repeat sequence
6. Autoradiography → produces a pattern of bands = the DNA "fingerprint"

• PCR amplifies multiple STR loci simultaneously (multiplex PCR)
• Fluorescently labeled primers
• Capillary electrophoresis separates fragments
• Computer generates the STR profile

• A child inherits one allele at each STR locus from each parent.
• Each STR allele in the child must match one allele from the mother or alleged father.
• Testing 13-16 STR loci simultaneously; if a match occurs at all loci, probability of paternity is >99.999%.
• If 3 or more loci show non-paternity, the alleged father is excluded.

• Criminal investigation (rape, murder - identifying suspect from trace evidence)
• Identification of disaster victims
• Establishing identity in immigration disputes
• Identifying remains of missing persons

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5. RFLP in Prenatal Diagnosis of Sickle Cell Anemia (2021)

• A point mutation (A→T at codon 6 of β-globin gene) in sickle cell disease abolishes a recognition site for the restriction enzyme MstII (recognition sequence: CCTNAGG).
• Normal β-globin: has MstII site (cut → two fragments)
• Sickle β-globin (HbS): mutation destroys the site (no cut → one larger fragment)

1. Obtain fetal DNA via chorionic villus sampling (CVS, 10-12 weeks) or amniocentesis (15-18 weeks)
2. Digest fetal and parental DNA with MstII restriction enzyme
3. Run gel electrophoresis
4. Southern blot and hybridize with β-globin probe
5. Analyze fragment pattern:
   • Normal (HbA/HbA): two bands (MstII cuts both alleles)
   • Sickle cell trait (HbA/HbS): three bands (one normal + one larger band)
   • Sickle cell disease (HbS/HbS): one large band only (neither allele is cut)

• Allows diagnosis before birth, giving parents the option of genetic counseling
• Does not require knowing the exact mutation - it is indirect diagnosis
• Can be applied to any disease linked to a restriction site change

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7. Discuss Briefly DNA Vector (2018)

• Ability to replicate autonomously in the host cell (must have an origin of replication)
• Restriction sites (Multiple Cloning Site/MCS) for insertion of foreign DNA
• Selectable marker (antibiotic resistance gene - e.g., ampicillin resistance) to identify transformed cells
• Moderate or high copy number
• Small size (to accommodate large inserts and easy manipulation)

• Cloning genes for study
• Production of recombinant proteins (insulin, erythropoietin, growth hormone, vaccines)
• Gene therapy vectors (viral vectors: adenovirus, AAV, lentivirus)

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8. Discuss Northern Blotting (2018, 2017)

1. RNA extraction: Total RNA or poly-A+ RNA (mRNA enriched) is isolated from cells/tissue under RNase-free conditions.
2. Gel electrophoresis: RNA is separated by size on a denaturing formaldehyde agarose gel (formaldehyde removes secondary structure, so RNA migrates by size alone).
3. Transfer: RNA is transferred to a nitrocellulose or nylon membrane (capillary or electroblotting).
4. UV crosslinking: RNA is fixed to the membrane.
5. Prehybridization: Block non-specific binding with salmon sperm DNA/BSA.
6. Hybridization: Labeled single-stranded probe (complementary to target mRNA) is added; hybridizes specifically with the target.
7. Washing: Remove non-specifically bound probe.
8. Detection: Autoradiography (if radioactive probe) or chemiluminescence.

• Presence of the specific mRNA
• Size of the mRNA (by position)
• Relative amount (by band intensity)

• Study gene expression in different tissues, development stages, or disease states
• Determine mRNA size and detect alternative splice variants
• Diagnose RNA virus infections
• Research in oncology (oncogene expression levels)

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9. Write a Short Note on Taq Polymerase (2016)

• Thermostability: Retains activity at 95°C (temperature used for DNA denaturation in PCR); not inactivated by repeated heating cycles
• Optimal temperature: 75-80°C (extension temperature in PCR = 72°C)
• 5'→3' polymerase activity: Extends primers in the 5'→3' direction
• 5'→3' exonuclease activity: Can degrade RNA/DNA ahead of it (used in TaqMan probes in real-time PCR)
• Lacks 3'→5' exonuclease (proofreading) activity: Error rate ~1 per 10⁵ bases (higher than proofreading polymerases like Pfu)
• Adds a non-templated 'A' overhang at the 3' end of PCR products (useful for TA cloning)
• Molecular weight: ~94 kDa

• Survives the denaturation step (94-96°C) repeatedly
• Synthesizes new DNA at 72°C, extending from primers

• Lacks proofreading activity → errors in PCR products (not suitable for cloning requiring high fidelity)
• For high-fidelity applications: Pfu polymerase (from Pyrococcus furiosus) or Phusion polymerase is preferred

• Standard PCR
• qPCR/real-time PCR (TaqMan assay uses Taq's 5'→3' exonuclease activity to cleave a fluorescent probe)
• RAPD, RFLP analysis

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