Each new double helix has one old strand and one new strand (proven by the Meselson-Stahl experiment using 15N 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 5โฒโ3โฒ (add nucleotides to the free 3โฒ-OH)
Antiparallel template: The template strand is read 3โฒโ5โฒ
Key Enzymes โ Complete Table
Enzyme
Function
Key Details
Helicase
Unwinds double helix
Breaks H-bonds between base pairs at the replication fork
Topoisomerase (Gyrase)
Relieves supercoiling
Cuts and rejoins DNA ahead of the fork to prevent tangling
SSB proteins
Keep strands separated
Bind single-stranded DNA to prevent re-annealing
Primase
Synthesizes RNA primer
Provides the free 3โฒ-OH that DNA Pol III needs to start
DNA Pol III
Main replication enzyme
synthesis + proofreading exonuclease
Leading vs. Lagging Strand
Leading strand: Template runs 3โฒโ5โฒ โ continuous synthesis toward the fork
Lagging strand: Template runs 5 โ discontinuous synthesis AWAY from the fork โ Okazaki fragments (~1000-2000 nt in bacteria, ~100-200 in eukaryotes)
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 3โฒ-OH) โ 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 โ cellular aging (Hayflick limit)
DNA Replication ๐ฏ
Eukaryotic vs. Prokaryotic Replication โ Comparison
Feature
Prokaryotes
Eukaryotes
Origins of replication
1 (OriC)
Many (~10,000 in human cells)
Speed
~1000 nt/sec
~50 nt/sec
Okazaki fragments
~1000-2000 nt
~100-200 nt
Main polymerase
DNA Pol III
DNA Pol delta (lagging) and epsilon (leading)
Topoisomerase
Gyrase (type II)
Topoisomerase I and II
Telomere issue
No (circular DNA)
Yes (linear chromosomes need telomerase)
Histones
No
Yes (old histones distributed to both daughter strands)
Drugs Targeting DNA Replication (MCAT Favorites!)
Drug
Target
Mechanism
Use
Fluoroquinolones (ciprofloxacin)
Bacterial gyrase
Blocks supercoil relief
Advanced Concepts ๐ฏ
Key Takeaways โ Part 1
Semiconservative replication proven by Meselson-Stahl (heavy isotope experiment)
DNA Pol III: 5โฒโ3โฒ synthesis, 3 proofreading โ needs a primer (3'-OH)
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 โ 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
Part 4: Gene Regulation
Molecular Biology for the MCAT
Part 4 of 7 โ 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 โ not different genes โ explains cell specialization. The MCAT tests regulation at every level.
Without tryptophan: Repressor inactive โ genes ON (cell makes tryptophan)
With tryptophan: Trp acts as corepressor โ binds repressor โ activates it โ repressor binds operator โ genes OFF
Also regulated by attenuation: Secondary structures in mRNA leader sequence cause premature termination when trp is abundant
Eukaryotic Gene Regulation โ Five Levels
Level
Mechanism
Effect
Example
Part 5: Mutations & Repair
Molecular Biology for the MCAT
Part 5 of 7 โ Mutations & DNA Repair
Types of Point Mutations
Mutation Type
What Happens
Effect on Protein
Example
Silent
New codon, SAME amino acid
None
GCU โ GCC (both = Ala)
Missense
New codon, DIFFERENT amino acid
Variable (conservative vs. non-conservative)
Sickle cell: Glu โ Val (GAG โ GUG)
Nonsense
Codon โ STOP codon
Truncated protein (usually nonfunctional)
Any โ UAA, UAG, or UGA
Conservative vs. non-conservative missense: Replacing an amino acid with a chemically similar one (conservative, e.g., Leu โ Ile) is less damaging than replacing with a dissimilar one (non-conservative, e.g., Glu โ Val in sickle cell disease).
Frameshift Mutations
Insertion or deletion of nucleotides NOT in multiples of 3 โ shifts entire reading frame
Every downstream codon is altered โ usually nonfunctional protein
Almost always more damaging than point mutations
Insertions/deletions in multiples of 3 โ add/remove amino acids WITHOUT shifting the frame
Transitions vs. Transversions
Part 6: Biotechnology & Lab Techniques
Molecular Biology for the MCAT
Part 6 of 7 โ Biotechnology Techniques
PCR (Polymerase Chain Reaction)
Amplifies a specific DNA sequence exponentially from a tiny sample.
Step
Temperature
What Happens
Duration
Denaturation
~95ยฐC
Double-stranded DNA separates into single strands
30 sec
Annealing
~55-65ยฐC
Short DNA primers bind (anneal) to complementary sequences flanking the target
30 sec
Extension
~72ยฐC
Taq polymerase synthesizes new DNA from primers
1-2 min
After n cycles: copies from one template molecule
Part 7: Review & MCAT Practice
Molecular Biology for the MCAT
Part 7 of 7 โ Viruses & Recombinant DNA
Virus Structure & Classification
Feature
DNA Viruses
RNA Viruses
Retroviruses
Genome
dsDNA usually
ssRNA or dsRNA
ssRNA
Replication
Host DNA Pol
RNA-dependent RNA Pol (RdRp)
Reverse transcriptase โ DNA โ integrase
Example
Herpes, Adenovirus
Influenza, Ebola
HIV
Viral Life Cycles
Lytic cycle: Virus replicates โ lyses host cell โ releases new virions
Lysogenic cycle: Viral DNA integrates into host genome (prophage) โ replicates with host โ can switch to lytic
Retroviruses (HIV)
5โฒโ3โฒ
3โฒโ5โฒ
DNA Pol I
Removes RNA primers
Replaces primers with DNA (5' โ 3' exonuclease removes primer)
Ligase
Joins Okazaki fragments
Seals phosphodiester backbone nicks
Sliding clamp (PCNA)
Processivity factor
Keeps DNA Pol III attached to the template
โฒ
โ
3โฒ
Each Okazaki fragment needs its own RNA primer
Antibiotic
AZT (zidovudine)
Reverse transcriptase
Chain terminator (nucleoside analog without 3'-OH)
HIV treatment
Methotrexate
Dihydrofolate reductase
Blocks thymidylate synthesis โ no dTTP
Cancer chemotherapy
Cytarabine
DNA polymerase
Nucleoside analog, inhibits chain elongation
Leukemia treatment
Repair After Replication โ Error Rates
DNA Pol III error rate: ~1 in 105 nucleotides (before proofreading)
After proofreading (3โฒโ5โฒ exonuclease): ~1 in 107
After mismatch repair: ~1 in 109 to 1010
This extraordinary accuracy is essential for genome stability
โฒ
โ
5โฒ
Leading strand: continuous. Lagging strand: discontinuous (Okazaki fragments, each with own primer)
Enzyme order: helicase โ topoisomerase (ahead) โ primase โ DNA Pol III โ DNA Pol I (removes primers) โ ligase
Telomeres shorten each division (end replication problem); telomerase extends them (active in stem/cancer cells)
Error rates: Pol III alone ~10โ5 โ with proofreading ~10โ7 โ with mismatch repair ~10โ9
Drugs: fluoroquinolones (gyrase), AZT (chain terminator for reverse transcriptase), methotrexate (dTTP synthesis)
Intron removal: precise cut at conserved GU (5' end of intron) and AG (3' end) sequences
Creates a lariat intermediate
Alternative Splicing โ One Gene, Multiple Proteins
Different combinations of exons โ different mRNAs โ 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 ๐ฏ
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 โ work through DNA looping
Mediator complex: Bridge between transcription factors and RNA Pol II
Inhibitors of Transcription โ MCAT Drug Connections
Inhibitor
Target
Clinical Use
Rifampin
Bacterial RNA polymerase
Tuberculosis treatment
Alpha-amanitin
Eukaryotic RNA Pol II
Mushroom poisoning (Amanita)
Actinomycin D
Intercalates DNA, blocks RNA Pol
Cancer chemotherapy
Key: Rifampin targets bacterial RNA Pol (one type) but NOT eukaryotic RNA Pol โ selective antibiotic. Alpha-amanitin is toxic to humans because it inhibits our RNA Pol II.
From DNA replication to gene regulation to virology โ molecular biology is the most heavily tested content on the MCAT Bio/Biochem section. Master the central dogma, regulation, and biotechnology techniques.