Comprehensive Notes on Gene Expression, Regulation, and Mutations

Transfer RNA (tRNA) and Ribosomal RNA (rRNA)

  • Transfer RNA (tRNA): Translates genetic code by bringing specific amino acids to ribosomes. It has a cloverleaf 2D shape, a 3D compact form, an anticodon complementary to mRNA, and an amino acid attachment site. Aminoacyl tRNA synthetases ensure correct amino acid attachment.

  • Ribosomal RNA (rRNA): Forms the structural and enzymatic core of ribosomes. It positions mRNA and tRNA during translation and forms peptide bonds (acting as a ribozyme).

  • Core Machinery: mRNA, tRNA, and rRNA are essential for protein synthesis.

The Genetic Code and Codons

  • Codon: A three-base sequence on mRNA that specifies an amino acid.

    • Anticodon: Complementary three-base sequence on tRNA.

    • Start Codon: AUG (methionine), signals translation start.

    • Stop Codons: UAA, UGA, UAG, signal translation end.

Features of the Genetic Code and Mutations

  • Degenerate Genetic Code: More than one codon can specify the same amino acid, providing a buffer against mutations.

  • Wobble Position: The third base of a codon is flexible in pairing, often tolerating mutations without changing the amino acid.

  • Silent Mutation: Nucleotide change that doesn't alter the amino acid sequence.

  • Point Mutations: Change in one nucleotide.

    • Missense: Changes codon to a different amino acid (e.g., sickle cell disease).

    • Nonsense: Changes codon to a stop codon, causing premature termination.

  • Frameshift Mutations: Insertion or deletion of nucleotides, shifting the reading frame and severely altering the protein, usually more disruptive than point mutations.

Transcription

  • Definition: Synthesizing an RNA molecule from a DNA template.

  • Purpose: Create a working RNA copy of a gene.

  • Steps:

    1. DNA Unwinding: Helicase separates strands; topoisomerase relieves tension.

    2. Template Strand: RNA polymerase reads the 3' to 5' DNA template (antisense) strand, synthesizing RNA in the 5' to 3' direction.

    3. Initiation: RNA polymerase binds to the promoter region (e.g., TATA Box in eukaryotes), aided by transcription factors.

    4. Eukaryotic RNA Polymerases:

      • RNA Pol I: rRNA

      • RNA Pol II: mRNA, snRNA

      • RNA Pol III: tRNA, some rRNA

    5. Elongation: RNA polymerase synthesizes heterogeneous nuclear RNA (hnRNA) at the +1 site.

Post-Transcriptional Processing (Eukaryotes)

  • Purpose: Convert hnRNA into stable, functional mRNA.

  • Three Major Modifications:

    1. Splicing: Removal of non-coding introns and joining of coding exons by the spliceosome (snRNAs + proteins).

    2. Five Prime Cap (5' Cap): Addition of 7-methylguanosine to the 5' end; aids ribosome recognition and prevents degradation.

    3. Three Prime Poly-A Tail (3' Polyadenylation): Addition of 100-250 adenine nucleotides to the 3' end; protects RNA and assists nuclear export.

  • Alternative Splicing: A single gene can produce multiple mRNA versions and proteins by differential splicing of exons, increasing protein diversity.

Translation

  • Location: Ribosomes in the cytoplasm.

  • Process: Reads mRNA to synthesize protein.

  • Components: mRNA, tRNA, amino acids, GTP.

  • Ribosome Structure: Large and small subunits (70S in prokaryotes, 80S in eukaryotes) with three tRNA binding sites:

    • A Site: Aminoacyl-tRNA entry.

    • P Site: Peptidyl-tRNA, holds growing chain.

    • E Site: Empty tRNA exit.

  • Steps:

    1. Initiation: Small subunit binds mRNA (Shine-Dalgarno in prokaryotes, 5' cap scan in eukaryotes), initiator tRNA (methionine) binds to AUG in P site, then large subunit joins.

    2. Elongation: New tRNA enters A site, peptidyltransferase forms peptide bond, ribosome translocates (A to P, P to E, E exits), repeating for each codon.

    3. Termination: Ribosome encounters stop codon (UAA, UAG, UGA), release factor binds, polypeptide is released, and ribosomal subunits dissociate.

  • Signal Sequences: Direct eukaryotic proteins to cellular locations.

Post-Translational Modifications

  • Purpose: Ensure proteins are fully functional.

  • Categories:

    1. Structural Changes: Cleavage (e.g., signal sequence removal), subunit assembly.

    2. Chemical Additions: Phosphorylation, carboxylation, glycosylation, prenylation (affecting activity, localization, or folding).

Gene Regulation in Prokaryotes: Operons

  • Operon: Group of related genes under a single promoter, increasing efficiency.

  • Components: Regulator gene (codes for repressor), promoter (RNA polymerase binding), operator (repressor binding), structural genes.

  • Types:

    1. Inducible Systems (e.g., Lac Operon):

      • Default OFF: Repressor binds operator.

      • Inducer (e.g., allolactose) removes repressor, turning genes ON.

      • Lac Operon also has positive control by cAMP-CAP when glucose is low, boosting transcription.

    2. Repressible Systems (e.g., Trp Operon):

      • Default ON: Repressor inactive.

      • Co-repressor (e.g., tryptophan) activates repressor, which binds operator and turns genes OFF (feedback inhibition).

Gene Regulation in Eukaryotes

  • Complexity: Multiple control points.

  • Mechanisms:

    1. Chromatin Structure:

      • Euchromatin: Loosely packed, active.

      • Heterochromatin: Densely packed, silent.

      • Histone Acetylation (HATs): Looses chromatin, increases transcription.

      • Histone Deacetylation (HDACs): Tightens chromatin, decreases transcription.

      • DNA Methylation: Adds methyl groups to DNA, long-term gene silencing.

    2. Transcription Factors: Proteins binding to promoters (near gene) and enhancers (far from gene) using DNA binding and activation domains. They can activate or repress transcription, often involving DNA looping.

    3. **Gene Amplification Mechanisms