Microbiology: DNA Replication, Gene Expression, Operons, and Mutations

DNA Replication and Chemotherapeutics

  1. Overview of DNA Replication

    • Purpose: Essential prior to cell division (binary fission in prokaryotes, mitosis in eukaryotes).

    • Mechanism: Semi-conservative replication, where each parental strand serves as a template to produce a new daughter strand.

    • Key Enzymes and Components:

    • Origin of Replication: AT-rich DNA region where strand separation initiates.

    • Helicase: Unwinds and opens the double-stranded DNA helix.

    • DNA Gyrase (Topoisomerase): Relieves supercoiling strain ahead of the replication fork by making temporary double-strand breaks and re-ligating them.

    • Single-Stranded Binding Proteins (SSBPs): Bind single-stranded DNA to prevent complementary bases from re-annealing.

    • Primase: Synthesizes short RNA primers to supply a free 3′-OH3'\text{-OH} group.

    • DNA Polymerase III: Main elongation enzyme; synthesizes DNA continuously or discontinuously in the 5′→3′5' \rightarrow 3' direction.

    • Leading vs. Lagging Strand:

      • Leading Strand: Synthesized continuously toward the replication fork.

      • Lagging Strand: Synthesized discontinuously away from the replication fork, creating Okazaki fragments.

    • DNA Polymerase I: Excisionally removes RNA primers on the lagging strand and fills gaps with DNA nucleotides.

    • DNA Ligase: Catalyzes phosphodiester bond formation to seal nicked backbones between Okazaki fragments.

  2. Chemotherapeutic Applications

    • Rapidly dividing cancer cells constantly replicate their DNA.

    • Mechanism: Chemotherapeutics targeting DNA gyrase/topoisomerase block supercoil relief, causing severe DNA strand breaks.

    • Cells accumulating unrepaired DNA breaks undergo apoptosis.

    • Pediatric Considerations: Young children have actively growing normal tissues, making topoisomerase inhibitors significantly more systemically toxic in pediatric oncology than in adults.

Gene Expression and Regulation

  1. Transcription (DNA to mRNA)

    • RNA Polymerase: Unwinds DNA and synthesizes mRNA 5′→3′5' \rightarrow 3' (reading the template strand 3′→5′3' \rightarrow 5').

    • Promoter Region: Upstream DNA sequence recruiting RNA polymerase.

    • Strong Promoter: High affinity for RNA polymerase; leads to high basal transcription.

    • Weak Promoter: Low affinity for RNA polymerase; enzyme frequently detaches, leading to low basal transcription.

    • Eukaryotic Control Elements:

    • Enhancers: DNA sequences binding activator proteins to recruit RNA polymerase and increase gene expression.

    • Silencers: DNA sequences binding repressor proteins to block RNA polymerase access and decrease gene expression.

  2. Prokaryotic vs. Eukaryotic Expression

    • Eukaryotes:

    • Spatial separation: Transcription occurs inside the nucleus; mRNA must be exported to the cytoplasm for translation.

    • Monocistronic: A single promoter regulates one gene, producing one protein product.

    • Prokaryotes:

    • Occurs in the nucleoid region of the cytoplasm.

    • Simultaneous Transcription and Translation: Ribosomes dock onto nascent mRNA while RNA polymerase is actively transcribing.

    • Polycistronic: A single promoter controls multiple clustered genes in an operon, yielding one polycistronic mRNA that codes for several proteins.

  3. Bacterial Operons

    • TRP Operon (Repressible Operon):

    • Encodes enzymes required for tryptophan biosynthesis.

    • Tryptophan Present: Tryptophan acts as a co-repressor, binding the repressor protein, which then binds the operator to block transcription.

    • Tryptophan Absent: Repressor remains inactive and unattached, allowing transcription of tryptophan synthesis enzymes.

    • LAC Operon (Inducible Operon):

    • Encodes enzymes for lactose catabolism (lacZlacZ, lacYlacY, lacAlacA).

    • E. coli prefers glucose over lactose.

    • Glucose High, Lactose Absent: Repressor bound to operator; transcription blocked.

    • Glucose High, Lactose High: Lactose binds repressor, removing it from operator. However, because the promoter is weak, transcription occurs at minimal levels.

    • Glucose Low, Lactose High: Low glucose elevates cyclic AMP (cAMP\text{cAMP}$). cAMP\text{cAMP} binds CAP to form the CAP-cAMP\text{CAP-cAMP} complex, which stabilizes RNA polymerase on the weak promoter, inducing high levels of transcription.

Translation, Mutations, and Viral Genetics

  1. Translation Mechanism

    • Ribosome: Translates mRNA 5′→3′5' \rightarrow 3', starting at the start codon (AUG\text{AUG}, coding for methionine).

    • tRNA: Delivers specific amino acids via complementary anticodon-codon pairing.

    • Synthesis: Catalyzes peptide bond formation through dehydration reactions from the N-terminus to the C-terminus (N→C\text{N} \rightarrow \text{C}$).

    • Termination: Stop codons (UAA\text{UAA}, UAG\text{UAG}, UGA\text{UGA}) recruit a release factor lacking an amino acid, triggering complex disassembly.

  2. Mutations and DNA Repair

    • Point Mutations / Base Substitutions:

    • Silent Mutation: Nucleotide change does not alter the amino acid sequence (often occurring at wobble positions).

    • Missense Mutation: Nucleotide change alters a codon to specify a different amino acid.

    • Nonsense Mutation: Converts an amino acid codon into a premature stop codon, truncating the polypeptide.

    • Frameshift Mutations: Insertion or deletion of bases (not in multiples of 3) alters the downstream reading frame, radically changing the protein sequence.

    • UV Damage & Repair:

    • UV light induces covalent bonding between adjacent pyrimidines, forming thymine dimers that distort DNA structure.

    • Nucleotide Excision Repair (NER): Enzyme complex that recognizes distortion, excises the damaged strand segment, and fills the gap using DNA polymerase and ligase.

    • Xeroderma Pigmentosum (XP): Autosomal recessive defect in NER genes, leading to extreme UV sensitivity, early lesions, and severe skin cancer risk.

  3. HIV Lifecycle and Genetic Resistance

    • Structure: Enveloped retrovirus containing two identical +ssRNA+\text{ssRNA} strands, a capsid, and glycoprotein spikes (GP120\text{GP120}).

    • Host Target: T-helper cells expressing the CD4\text{CD4} surface receptor.

    • Entry Mechanism:

    1. GP120\text{GP120} binds CD4\text{CD4}.

    2. Structural shift allows secondary binding to co-receptor (CCR5\text{CCR5} or CXCR4\text{CXCR4}).

    3. Viral envelope fuses with host plasma membrane, releasing the capsid inside.

    • CCR5-delta 32 Mutation:

    • A 32-base pair32\text{-base pair} deletion in the CCR5\text{CCR5} gene produces a truncated co-receptor.

    • Prevents membrane fusion and confers immunity against HIV-1 r5\text{HIV-1 r5} strains (though individuals remain susceptible to HIV-1 x4\text{HIV-1 x4} strains using CXCR4\text{CXCR4}).