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