Master Molecular Biology
for USMLE Step 1

Core Concepts

Molecular biology underpins all biological processes. Key concepts include:

• DNA Structure: Double helix, antiparallel strands (5' to 3' and 3' to 5'), phosphodiester backbone, complementary base pairing (A-T, G-C). Stabilized by H-bonds. Supercoiling managed by topoisomerases.
• DNA Replication (S Phase): Semiconservative process.
  • Helicase: Unwinds DNA. SSBPs: Prevent reannealing.
  • Primase: Synthesizes RNA primers.
  • DNA Polymerase III (prokaryotes) / DNA Pol δ and ε (eukaryotes): Synthesizes new DNA in 5'->3' direction. Has 3'->5' exonuclease (proofreading) activity.
  • Leading Strand: Continuous synthesis. Lagging Strand: Discontinuous, forms Okazaki fragments.
  • DNA Polymerase I (prokaryotes) / RNase H & DNA Pol α (eukaryotes): Removes RNA primers. DNA Pol I also has 5'->3' exonuclease activity (repair).
  • DNA Ligase: Joins Okazaki fragments.
  • Telomeres & Telomerase: Non-coding ends of chromosomes. Telomerase (reverse transcriptase) adds TTAGGG repeats, active in stem cells & cancer, protects genetic info.
• Transcription (DNA to RNA):
  • RNA Polymerases: I (rRNA), II (mRNA, snRNA, miRNA), III (tRNA, 5S rRNA).
  • Promoters: TATA box. Transcription factors bind to regulate.
  • Post-transcriptional Modifications (eukaryotes only):
    • 5' Capping: 7-methylguanosine cap, protects from degradation, essential for translation initiation.
    • 3' Polyadenylation: Poly-A tail (~200 A's), protects from degradation, aids in export & translation.
    • Splicing: snRNPs form spliceosome to remove introns (non-coding) and ligate exons (coding).
• Translation (RNA to Protein):
  • Genetic Code: Universal, degenerate (multiple codons for one AA), unambiguous. Start codon: AUG (Methionine). Stop codons: UAA, UAG, UGA.
  • tRNA: Has anticodon, carries specific amino acid. Aminoacyl-tRNA synthetases attach correct AA (requires ATP).
  • Ribosomes: Prokaryotes (30S+50S = 70S); Eukaryotes (40S+60S = 80S).
  • Stages: Initiation (mRNA, initiator tRNA, ribosomal subunits assemble), Elongation (A site for incoming tRNA, P site for peptidyl tRNA, E site for exiting tRNA), Termination (release factors bind to stop codon).
  • Post-translational Modifications: Folding (chaperones), cleavage, glycosylation, phosphorylation, ubiquitination.
• DNA Repair Mechanisms:
  • Mismatch Repair (MMR): Corrects replication errors (e.g., G-T mismatch).
  • Nucleotide Excision Repair (NER): Repairs bulky lesions (e.g., pyrimidine dimers from UV).
  • Base Excision Repair (BER): Repairs single damaged base (e.g., deamination) via glycosylase.
  • Non-Homologous End Joining (NHEJ): Repairs double-strand breaks by ligating ends, often error-prone.
  • Homologous Recombination (HR): Repairs double-strand breaks using sister chromatid as template, error-free.
• Gene Regulation: Prokaryotic operons (e.g., Lac/Trp). Eukaryotic gene regulation involves transcription factors, enhancers/silencers, chromatin remodeling (histone acetylation/methylation), and DNA methylation (gene silencing).
• Mendelian & Non-Mendelian Genetics: Autosomal dominant/recessive, X-linked inheritance, mitochondrial inheritance (maternal), trinucleotide repeat disorders (anticipation), imprinting. Hardy-Weinberg equilibrium.

Clinical Presentation (Molecular Defects)

• Genetic Disorders: Caused by mutations (point, frameshift, deletion/insertion, trinucleotide repeats) in specific genes. Examples: Cystic Fibrosis (CFTR), Sickle Cell Anemia (HbS), Huntington's disease (HTT), Fragile X syndrome (FMR1).
• Cancers: Often due to accumulation of mutations in proto-oncogenes (gain of function) and tumor suppressor genes (loss of function), and/or defects in DNA repair pathways.
• Mitochondrial Diseases: Result from mutations in mitochondrial DNA (maternally inherited) or nuclear DNA encoding mitochondrial proteins, leading to energy deficits.
• Infectious Diseases: Many pathogens (viruses, bacteria) exploit or target host molecular machinery for replication or pathogenesis.

Diagnosis (Molecular Tools)

Diagnosis of molecular defects often relies on specific techniques:

• PCR (Polymerase Chain Reaction): Amplifies specific DNA sequences. Used for pathogen detection, genetic screening.
• RT-PCR (Reverse Transcriptase PCR): Converts RNA to cDNA, then amplifies. Used for gene expression analysis, viral load (e.g., HIV, HCV).
• Southern Blot: Detects specific DNA sequences in a sample. Used for gene deletions/rearrangements (e.g., sickle cell, trinucleotide repeats).
• Northern Blot: Detects specific RNA sequences. Used for gene expression levels.
• Western Blot: Detects specific proteins. Used for protein presence, size, and post-translational modifications.
• ELISA (Enzyme-Linked Immunosorbent Assay): Detects and quantifies proteins (antigens or antibodies).
• FISH (Fluorescence In Situ Hybridization): Uses fluorescent probes to detect specific DNA sequences on chromosomes. Used for microdeletions, translocations (e.g., DiGeorge, CML).
• DNA Sequencing (Sanger, Next-Gen): Determines exact nucleotide sequence. Gold standard for identifying specific point mutations, small indels, and large-scale genetic variations.
• Karyotyping: Visualizes and analyzes chromosome number and gross structure (e.g., Down Syndrome).

Management (Therapeutic Implications)

Understanding molecular biology guides targeted therapies:

• Gene Therapy: Introducing, inactivating, or replacing a gene to treat disease (e.g., severe combined immunodeficiency, spinal muscular atrophy).
• Enzyme Replacement Therapy: Providing functional enzymes missing due to genetic defects (e.g., lysosomal storage diseases).
• Targeted Drugs: Inhibitors designed to target specific proteins or pathways (e.g., tyrosine kinase inhibitors for oncogenes like BCR-ABL in CML; drugs affecting DNA replication/transcription in cancer chemotherapy).
• Antimicrobials: Many antibiotics target unique prokaryotic molecular machinery (e.g., ribosomal inhibitors, DNA gyrase inhibitors). Antivirals target specific viral enzymes (e.g., reverse transcriptase inhibitors, protease inhibitors).
• siRNA/miRNA Therapy: Using small RNA molecules to silence specific gene expression.