Molecular Biology

Molecular Biology for the MCAT

Part 1 of 7 — DNA Replication

DNA Replication is Semiconservative

Each new double helix has one old strand and one new strand (proven by the Meselson-Stahl experiment using $^{15}$N heavy isotope labeling). After one round, all molecules were intermediate density. After two rounds, half intermediate and half light.

Replication Overview

• Bidirectional: Proceeds in both directions from each origin of replication
• Origins: Bacteria have one origin; eukaryotes have many (to speed up replication of much larger genomes)
• Direction: DNA polymerases can ONLY synthesize $5' \to 3'$ (add nucleotides to the free $3'$-OH)
• Antiparallel template: The template strand is read $3' \to 5'$

Key Enzymes — Complete Table

Leading vs. Lagging Strand

• Leading strand: Template runs $3' \to 5'$ → continuous synthesis toward the fork
• Lagging strand: Template runs $5' \to 3'$ → discontinuous synthesis AWAY from the fork → Okazaki fragments (~1000-2000 nt in bacteria, ~100-200 in eukaryotes)
• Each Okazaki fragment needs its own RNA primer

Telomeres and Telomerase

• Telomeres: Protective caps at chromosome ends (TTAGGG repeats in humans)
• End replication problem: When the last RNA primer on the lagging strand is removed, DNA Pol cannot fill the gap (no upstream $3'$-OH) → telomere shortens each division
• Telomerase: Reverse transcriptase that uses an internal RNA template to extend telomeres
• Active in: stem cells, germ cells, cancer cells
• Inactive in: most somatic cells → cellular aging (Hayflick limit)

DNA Replication 🎯

Eukaryotic vs. Prokaryotic Replication — Comparison

Drugs Targeting DNA Replication (MCAT Favorites!)

Repair After Replication — Error Rates

• DNA Pol III error rate: ~1 in $10^5$ nucleotides (before proofreading)
• After proofreading ($3' \to 5'$ exonuclease): ~1 in $10^7$
• After mismatch repair: ~1 in $10^9$ to $10^{10}$
• This extraordinary accuracy is essential for genome stability

Advanced Concepts 🎯

Key Takeaways — Part 1

• Semiconservative replication proven by Meselson-Stahl (heavy isotope experiment)
• DNA Pol III: $5' \to 3'$ synthesis, $3' \to 5'$ proofreading — needs a primer (3'-OH)
• Leading strand: continuous. Lagging strand: discontinuous (Okazaki fragments, each with own primer)
• Enzyme order: helicase → topoisomerase (ahead) → primase → DNA Pol III → DNA Pol I (removes primers) → ligase
• Telomeres shorten each division (end replication problem); telomerase extends them (active in stem/cancer cells)
• Error rates: Pol III alone ~$10^{-5}$ → with proofreading ~$10^{-7}$ → with mismatch repair ~$10^{-9}$
• Drugs: fluoroquinolones (gyrase), AZT (chain terminator for reverse transcriptase), methotrexate (dTTP synthesis)

Molecular Biology for the MCAT

Part 2 of 7 — Transcription

The Central Dogma

$\text{DNA} \xrightarrow{\text{Transcription}} \text{RNA} \xrightarrow{\text{Translation}} \text{Protein}$

RNA Polymerase — The Key Enzyme

• Reads template strand $3' \to 5'$, synthesizes mRNA $5' \to 3'$
• Does NOT need a primer (unlike DNA polymerase)
• Does NOT have proofreading ability → higher error rate than DNA replication (acceptable because mRNA is temporary)
• The mRNA sequence matches the coding strand (non-template), except U replaces T

Prokaryotic vs. Eukaryotic Transcription

Eukaryotic mRNA Processing — Three Essential Steps

1. 5' Cap (7-methylguanosine)

• Added co-transcriptionally (while transcription is ongoing)
• Functions: protects from 5' exonucleases, recognized by ribosome for translation initiation, aids nuclear export

2. 3' Poly-A Tail (~200 adenines)

• Added by poly-A polymerase after cleavage at AAUAAA signal
• Functions: protects from 3' exonucleases, facilitates nuclear export, aids translation

3. Splicing (by the spliceosome)

• Removes introns, joins exons
• Spliceosome = snRNPs (small nuclear ribonucleoproteins) + snRNAs
• Intron removal: precise cut at conserved GU (5' end of intron) and AG (3' end) sequences
• Creates a lariat intermediate

Alternative Splicing — One Gene, Multiple Proteins

• Different combinations of exons → different mRNAs → different proteins from ONE gene
• Explains how ~20,000 human genes can produce >100,000 proteins
• Example: Drosophila DSCAM gene can produce >38,000 different mRNAs!

Transcription 🎯

Transcription Factors and Enhancers

• General transcription factors (TFIIA, TFIIB, TFIID, etc.): Required for ALL Pol II genes. TFIID contains TBP (TATA-binding protein) that recognizes the TATA box.
• Specific transcription factors (activators/repressors): Bind enhancers or silencers to modulate transcription rate
• Enhancers: Can be thousands of base pairs upstream or downstream of the gene — work through DNA looping
• Mediator complex: Bridge between transcription factors and RNA Pol II

Inhibitors of Transcription — MCAT Drug Connections

Key: Rifampin targets bacterial RNA Pol (one type) but NOT eukaryotic RNA Pol → selective antibiotic. Alpha-amanitin is toxic to humans because it inhibits our RNA Pol II.

The mRNA Lifecycle

$\text{Transcription} \to \text{Processing (cap, tail, splice)} \to \text{Export through nuclear pore} \to \text{Translation} \to \text{Degradation}$

• mRNA stability varies: some last minutes (growth factor mRNAs), others last days (globin mRNA)
• AU-rich elements (AREs) in 3' UTR mark mRNA for rapid degradation
• microRNAs (miRNAs) can target specific mRNAs for degradation or translational repression

Advanced Transcription 🎯

Key Takeaways — Part 2

• RNA Pol II transcribes mRNA in eukaryotes; reads template $3' \to 5'$, synthesizes $5' \to 3'$; no primer needed
• Eukaryotic mRNA processing: 5' cap (protection + ribosome recognition) + poly-A tail (stability) + splicing (intron removal)
• Splicing by spliceosome at GU---AG junctions; introns form lariat intermediate
• Alternative splicing: one gene → multiple proteins (explains protein diversity)
• Prokaryotes: no mRNA processing, transcription-translation coupled (no nuclear envelope)
• Drugs: rifampin (bacterial RNA Pol), alpha-amanitin (eukaryotic RNA Pol II), actinomycin D (intercalation)
• mRNA stability regulated by 3' UTR elements, poly-A tail length, and miRNAs

Molecular Biology for the MCAT

Part 3 of 7 — Translation (Protein Synthesis)

The Genetic Code — Properties

• 64 codons = 4 bases in groups of 3 ($4^3 = 64$)
• 61 sense codons (amino acids) + 3 stop codons (UAA, UAG, UGA)
• Start codon: AUG = methionine (also signals ribosome binding in eukaryotes; fMet in prokaryotes)
• Degenerate (redundant): Multiple codons per amino acid (especially at 3rd "wobble" position)
• NOT ambiguous: Each codon specifies exactly ONE amino acid
• Universal (nearly): Same code in almost all organisms (minor exceptions in mitochondria)

Wobble Position — Why Degeneracy Matters

The 3rd base of a codon has "relaxed" base-pairing rules:

• One tRNA can recognize multiple codons that differ only at position 3
• This is why most synonymous mutations (silent) occur at position 3
• Wobble pairing: G-U is allowed at position 3 (not normally allowed elsewhere)

Ribosome Structure and Sites

Ribosome sites (on large subunit):

Translation Steps — Initiation, Elongation, Termination

Initiation (rate-limiting step):

• Prokaryotes: 30S binds Shine-Dalgarno sequence on mRNA → finds AUG → fMet-tRNA in P site → 50S joins
• Eukaryotes: 40S + initiator Met-tRNA binds 5' cap → scans for first AUG (Kozak sequence context) → 60S joins

Elongation (cyclical):

1. Charged tRNA enters A site (requires EF-Tu + GTP in prokaryotes)
2. Peptidyl transferase forms peptide bond (23S rRNA = ribozyme!)
3. Translocation: ribosome moves one codon toward 3' end (requires EF-G + GTP)
4. tRNA shifts P → E; A site open for next tRNA

Termination:

• Stop codon (UAA, UAG, UGA) enters A site
• Release factor binds (mimics tRNA shape) → peptide released → ribosome dissociates

Translation 🎯

Post-Translational Modifications

After translation, proteins must be properly modified and folded:

Antibiotics Targeting Translation — MCAT Must-Know

Aminoacyl-tRNA Synthetase — The Second Genetic Code

• 20 aminoacyl-tRNA synthetases (one per amino acid)
• Charges tRNA: amino acid + tRNA + ATP → aminoacyl-tRNA + AMP + PPi
• Recognition: enzyme recognizes BOTH the amino acid AND specific features of the tRNA (acceptor stem, anticodon loop)
• Proofreading (editing site): hydrolyzes incorrectly attached amino acids
• This is called the "second genetic code" because accuracy of translation depends on correct charging

Advanced Translation 🎯

Key Takeaways — Part 3

• Genetic code: 64 codons, degenerate but NOT ambiguous, nearly universal. Start = AUG, Stop = UAA/UAG/UGA
• Wobble at 3rd position explains degeneracy and why silent mutations cluster there
• Ribosome sites: A (entry), P (peptide), E (exit). Peptidyl transferase = ribozyme (rRNA catalysis)
• Prokaryotic initiation: Shine-Dalgarno. Eukaryotic: 5' cap scanning for AUG in Kozak context
• Aminoacyl-tRNA synthetases = "second genetic code" — charge tRNAs with correct amino acids
• Antibiotics: 30S targets (tetracycline, aminoglycosides) vs. 50S targets (chloramphenicol, erythromycin, clindamycin)
• Toxins: diphtheria (EF-2 ADP-ribosylation), puromycin (premature termination)

Molecular Biology for the MCAT

Part 4 of 7 — Gene Regulation

Why Gene Regulation Matters

Every cell has the same DNA, but a neuron looks and acts nothing like a liver cell. Differential gene expression — not different genes — explains cell specialization. The MCAT tests regulation at every level.

Prokaryotic Gene Regulation: The Operon Model

Lac Operon (inducible — normally OFF):

• Structural genes: lacZ (beta-galactosidase), lacY (permease), lacA (transacetylase)
• Without lactose: Repressor (lacI product) binds operator → blocks RNA Pol → genes OFF
• With lactose: Allolactose (isomer of lactose) binds repressor → conformational change → repressor falls off → genes ON
• Dual control: Low glucose → high cAMP → cAMP-CAP binds promoter → enhanced transcription
• Maximum expression: lactose present (repressor off) + glucose absent (cAMP-CAP active)

Trp Operon (repressible — normally ON):

• Without tryptophan: Repressor inactive → genes ON (cell makes tryptophan)
• With tryptophan: Trp acts as corepressor → binds repressor → activates it → repressor binds operator → genes OFF
• Also regulated by attenuation: Secondary structures in mRNA leader sequence cause premature termination when trp is abundant

Eukaryotic Gene Regulation — Five Levels

Epigenetics — HIGH YIELD

Key enzymes: HATs (histone acetyltransferases) = activate. HDACs (histone deacetylases) = silence. HDAC inhibitors are used as cancer drugs.

Gene Regulation 🎯

microRNA (miRNA) and siRNA — Post-Transcriptional Silencing

• miRNA: Endogenous ~22 nt RNAs that bind complementary sequences in 3' UTR of target mRNAs
  • Partial complementarity → translational repression (mRNA not translated)
  • High complementarity → mRNA degradation
  • RISC complex (RNA-induced silencing complex) mediates the effect
• siRNA: Exogenous or synthetic small RNAs → same RISC pathway → mRNA degradation
• Both are mechanisms of RNA interference (RNAi) — a major research tool and potential therapy

X-Inactivation (Barr Body)

• In females (XX), one X chromosome is randomly inactivated in each cell → Barr body (dense heterochromatin)
• XIST RNA: Long non-coding RNA that coats the inactive X → recruits silencing complexes
• Results in dosage compensation (males and females express ~same amount of X-linked genes)
• Random inactivation → mosaicism (e.g., calico cats, manifesting carriers of X-linked diseases)

Genomic Imprinting

• Some genes are expressed from only ONE parental allele (the other is silenced by methylation)
• Imprinting is parent-of-origin specific: e.g., IGF2 expressed from paternal allele only
• Deletion of the active allele → disease (even though the other allele is intact, it is silenced)
• Prader-Willi syndrome: paternal deletion at 15q11-13
• Angelman syndrome: maternal deletion at the SAME region (different genes affected)

Epigenetics and Cancer

• Cancer cells often show global hypomethylation (genome-wide) + local hypermethylation (at tumor suppressor promoters)
• Hypomethylation → genomic instability, oncogene activation
• Hypermethylation at CpG islands → tumor suppressor silencing (e.g., BRCA1, p16 promoter methylation)

Advanced Regulation 🎯

Key Takeaways — Part 4

• Lac operon: inducible (normally OFF); max expression = lactose ON + glucose OFF (high cAMP-CAP)
• Trp operon: repressible (normally ON); trp = corepressor that activates the repressor
• Eukaryotic regulation: epigenetic → transcriptional → post-transcriptional → translational → post-translational
• Epigenetics: DNA methylation = silencing; histone acetylation = activation; HDAC inhibitors = cancer therapy
• miRNA/siRNA: post-transcriptional silencing via RISC complex (RNAi)
• X-inactivation: random, XIST RNA-mediated → Barr body → mosaicism in females
• Genomic imprinting: parent-of-origin allele silencing (Prader-Willi vs. Angelman)
• Cancer epigenetics: global hypomethylation + local hypermethylation at tumor suppressor promoters

Molecular Biology for the MCAT

Part 5 of 7 — Mutations & DNA Repair

Types of Point Mutations

Conservative vs. non-conservative missense: Replacing an amino acid with a chemically similar one (conservative, e.g., Leu → Ile) is less damaging than replacing with a dissimilar one (non-conservative, e.g., Glu → Val in sickle cell disease).

Frameshift Mutations

• Insertion or deletion of nucleotides NOT in multiples of 3 → shifts entire reading frame
• Every downstream codon is altered → usually nonfunctional protein
• Almost always more damaging than point mutations
• Insertions/deletions in multiples of 3 → add/remove amino acids WITHOUT shifting the frame

Transitions vs. Transversions

DNA Repair Mechanisms — Complete Overview

Mutations & Repair 🎯

Mutagens — Agents That Cause Mutations

Ames Test — Detecting Mutagens

• Uses Salmonella bacteria that cannot synthesize histidine (his$^-$ mutant)
• Expose to suspected mutagen → plate on histidine-free media
• If colonies grow → reversion mutations occurred → substance is a mutagen (likely carcinogen)
• More colonies = more mutagenic

Nonsense-Mediated Decay (NMD)

• Quality control mechanism that degrades mRNAs with premature stop codons
• Prevents translation of truncated, potentially harmful proteins
• If a stop codon appears >50 nt upstream of the last exon-exon junction → mRNA degraded
• Clinically important: some genetic diseases are caused by NMD destroying mRNA before any protein is made

p53 — The Guardian of the Genome

p53 is the central hub connecting DNA damage to cell fate:

1. DNA damage detected → ATM/ATR kinases activate → phosphorylate p53 → stabilize it (normally degraded by MDM2)
2. p53 activates p21 (CDK inhibitor) → cell cycle arrest at G$_1$/S
3. If damage is repairable → DNA repair occurs → cell cycle resumes
4. If damage is irreparable → p53 activates Bax → apoptosis
5. p53 also upregulates DNA repair genes

p53 is mutated or inactivated in >50% of all human cancers — the single most commonly altered gene in cancer.

Advanced Mutations 🎯

Key Takeaways — Part 5

• Point mutations: silent (no change) < conservative missense < non-conservative missense < nonsense (truncation)
• Frameshifts (insertions/deletions not in multiples of 3): most devastating, alter all downstream codons
• Transitions (purine↔purine, pyrimidine↔pyrimidine) more common than transversions
• Repair hierarchy: proofreading → mismatch repair (Lynch syndrome) → BER (small damage) → NER (bulky lesions, XP)
• Double-strand break repair: HR (accurate, needs sister chromatid, BRCA1/2) vs. NHEJ (error-prone, any phase)
• Ames test: his$^-$ Salmonella reversion on mutagen exposure = carcinogen screen
• p53: DNA damage → cell cycle arrest (p21) or apoptosis (Bax); mutated in >50% of cancers

Molecular Biology for the MCAT

Part 6 of 7 — Biotechnology Techniques

PCR (Polymerase Chain Reaction)

Amplifies a specific DNA sequence exponentially from a tiny sample.

• After $n$ cycles: $2^n$ copies from one template molecule
• After 30 cycles: ~$10^9$ copies (1 billion)!
• Taq polymerase: From Thermus aquaticus (thermophilic bacterium) — survives 95°C denaturation

Requirements: Template DNA + two primers + dNTPs + Taq polymerase + Mg$^{2+}$

Gel Electrophoresis

• DNA migrates toward the positive electrode (anode) because DNA is negatively charged (phosphate backbone)
• Smaller fragments travel faster (farther from wells)
• Agarose gels: Separate large fragments (100 bp - 50 kb). PAGE: Separate small fragments or proteins
• Stain with ethidium bromide (DNA) or Coomassie blue (protein)

Blotting Techniques — "SNoW DRoP" Mnemonic

Southern = DNA, Northern = RNA, Western = Protein → SNoW DRoP

Restriction Enzymes

• Cut DNA at specific palindromic sequences (read same on both strands 5' → 3')
• Example: EcoRI recognizes GAATTC and cuts between G and A on each strand
• Sticky ends: Overhang (easier to ligate) vs. Blunt ends: Straight cut
• Used in molecular cloning, RFLP analysis, and DNA fingerprinting

Biotechnology 🎯

Molecular Cloning

1. Cut target DNA and vector (plasmid) with the same restriction enzyme → compatible sticky ends
2. Ligate: DNA ligase joins insert into the vector
3. Transform: Introduce recombinant plasmid into bacteria (heat shock or electroporation)
4. Select: Use antibiotic resistance gene on plasmid → only transformed bacteria survive
5. Screen: Blue-white screening (lacZ disruption), colony PCR, or sequencing

CRISPR-Cas9 — Precise Gene Editing

• CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats (bacterial immune defense)
• Guide RNA (sgRNA): 20-nt sequence complementary to target DNA
• Cas9: Nuclease that creates a double-strand break at the target site
• Repair outcomes: NHEJ → gene knockout (insertions/deletions disrupt gene) or HDR → precise gene insertion (if template provided)
• Applications: disease gene correction, cancer immunotherapy (CAR-T cells), agricultural biotechnology

Other Key Techniques

Advanced Techniques 🎯

Key Takeaways — Part 6

• PCR: Denature (95°C) → Anneal (~55°C) → Extend (72°C). $2^n$ copies after $n$ cycles. Taq polymerase is heat-stable but lacks proofreading.
• Gel electrophoresis: DNA runs to positive electrode; smaller fragments migrate farther
• Blots: Southern (DNA), Northern (RNA), Western (Protein) — "SNoW DRoP"
• Restriction enzymes cut palindromic sequences; sticky ends are easier to ligate than blunt ends
• Molecular cloning: cut → ligate → transform → select → screen
• CRISPR-Cas9: guide RNA + Cas9 nuclease → DSB → NHEJ (knockout) or HDR (precise edit)
• RT-PCR detects mRNA (via cDNA); Sanger sequencing uses ddNTP chain terminators
• Know when to use each technique for MCAT experimental design passages

Molecular Biology for the MCAT

Part 7 of 7 — Viruses & Recombinant DNA

Virus Structure & Classification

Viral Life Cycles

Lytic cycle: the virus replicates, lyses the host cell, and releases new virions. Lysogenic cycle: the viral DNA integrates into the host genome (a prophage), replicates passively with the host, and can later switch to the lytic cycle.

Retroviruses (HIV)

$\text{ssRNA} \xrightarrow{\text{reverse transcriptase}} \text{dsDNA} \xrightarrow{\text{integrase}} \text{provirus (in host DNA)}$

Reverse transcriptase lacks proofreading ($3' \to 5'$ exonuclease) activity, giving HIV a very high mutation rate.

Prions

• NOT viruses — they are misfolded proteins (no nucleic acid at all)
• They convert normally folded proteins into the misfolded form (a chain reaction)
• Resistant to standard sterilization (heat, UV, proteases)
• Examples: mad cow disease (BSE), Creutzfeldt-Jakob disease

Recombinant DNA — The Cloning Toolkit

Worked Example — Designing a Recombinant Insulin Plasmid

Problem. You want bacteria (E. coli) to produce human insulin. You have the human insulin mRNA, a plasmid with an ampicillin-resistance gene, restriction enzymes, ligase, and reverse transcriptase. Outline the cloning strategy and explain two MCAT-classic pitfalls.

Step 1 — Make cDNA, not genomic DNA. Use reverse transcriptase on the insulin mRNA to synthesize complementary DNA (cDNA). Pitfall #1: if you cloned the genomic gene instead, it would contain introns, and bacteria cannot splice (they lack the spliceosome), so they could not produce functional insulin. cDNA is already intron-free.

Step 2 — Cut insert and vector with the SAME restriction enzyme. Using one enzyme on both the cDNA and the plasmid generates complementary sticky ends, so the insert base-pairs with the opened vector. Pitfall #2: you must use compatible ends — mismatched ends won't anneal.

Step 3 — Ligate. DNA ligase seals the phosphodiester backbone, covalently inserting the gene into the plasmid to form recombinant DNA.

Step 4 — Transform and select. Introduce plasmids into E. coli (transformation), then plate on ampicillin. Only bacteria that took up the plasmid (carrying the ampicillin-resistance marker) survive — this is the selectable marker doing its job.

Step 5 — Express. A promoter on the plasmid drives transcription and translation of the insulin gene.

Conclusion. Reverse-transcribe mRNA → cut insert + vector with the same enzyme → ligate → transform → select with antibiotic → express. The two high-yield ideas: use cDNA to avoid introns, and use a selectable marker to find successful transformants.

Viruses & Biotech 🎯

Molecular Biology — Complete! ✅

From DNA replication to gene regulation to virology and biotechnology, molecular biology is the most heavily tested content on the MCAT Bio/Biochem section. Master the central dogma, regulation, and biotechnology techniques.

High-yield reminders:

• Retroviruses: ssRNA → (reverse transcriptase) → dsDNA → (integrase) → provirus; no proofreading = rapid mutation
• Lytic = lyse now; lysogenic = integrate as a prophage and wait
• Prions are infectious misfolded proteins with no nucleic acid
• Clone with cDNA (no introns) and select transformants with an antibiotic-resistance marker