Protein Metabolism

Codon Degeneracy vs. Wobble Pairing:

Codon Degeneracy: • Definition: Multiple different codons can specify the same amino acid • Pattern: 61 sense codons encode 20 amino acids • Distribution: Varies by amino acid (Met, Trp = 1 codon; Leu, Ser, Arg = 6 codons each) • Impact: Provides redundancy in the genetic code, allowing mutations without changing amino acid sequence

Wobble Pairing: • Definition: Flexible base-pairing between the 3rd codon position and 1st anticodon position • Mechanism: Less stringent hydrogen bonding at these positions • Function: Allows a single tRNA to recognize multiple codons • Special case: Inosine (I) in anticodon can pair with U, C, or A in codon

Relationship: • Wobble pairing is the physical mechanism that accommodates codon degeneracy • Reduces the number of tRNAs needed (fewer than 61 tRNAs required) • Most degeneracy occurs at the 3rd codon position, precisely where wobble occurs • Together they provide flexibility in translation while maintaining accuracy

Example: The four GNN codons (GUU, GUC, GUA, GUG) all specify valine and might be recognized by fewer than four different tRNAs due to wobble.

mRNA editing refers to post-transcriptional modifications that alter mRNA sequences before translation:

• Types of editing:

1. Nucleotide insertion/deletion
   • Example: Insertion of four U residues in cytochrome oxidase subunit II mRNA in protist mitochondria
   • Function: Shifts reading frame to produce correct protein
2. Base conversion
   • Example: C-to-U deamination in apolipoprotein B mRNA (changes CAA→UAA)
   • Function: Creates tissue-specific protein variants (apoB-100 in liver vs. apoB-48 in intestine)

• Mechanisms:

• Guide RNAs may serve as templates for editing (in trypanosomes)
• Specific enzymes (like cytidine deaminase) modify targeted bases
• Often involve recognition of specific RNA structures or sequences

• Significance for proteome diversity:

1. Allows synthesis of multiple proteins from single gene
2. Enables tissue-specific protein expression
3. Provides regulatory control at post-transcriptional level
4. Creates protein variants without requiring separate genes
5. Expands functional capacity of genome without increasing size

• Evolutionary advantage: Increases proteome complexity while maintaining genomic economy

The four key structural arms of tRNA are:

1. Amino acid arm: Located at the 3' end with the terminal CCA sequence; directly participates in amino acid attachment via esterification of the amino acid to the 3' hydroxyl of the terminal adenosine

2. TΨC arm: Contains ribothymidine (T) and pseudouridine (Ψ); interacts with the large ribosomal subunit during translation

3. D arm: Contains dihydrouridine (D) residues; contributes to the overall folding of tRNA

4. Anticodon arm: Contains the anticodon triplet that base-pairs with the mRNA codon

The amino acid arm is the direct site of aminoacylation, creating the critical link between an amino acid and its corresponding genetic code information.

Energy expenditure per amino acid:

1. Aminoacyl-tRNA formation:

   • ATP → AMP + PPi (equivalent to 2 ATP)
   • Additional ATP for proofreading (when incorrect amino acid is hydrolyzed)

2. Elongation cycle:

   • EF-Tu binding and delivery: 1 GTP → GDP + Pi
   • Translocation: 1 GTP → GDP + Pi

Total minimum energy cost: 4 high-energy phosphate bonds (>4 when proofreading occurs)

Thermodynamic analysis:

• Energy from 4 NTP→NDP: 4 × 30.5 kJ/mol = 122 kJ/mol
• Energy of peptide bond formation: -21 kJ/mol
• Net free-energy change: ~101 kJ/mol

Significance: This represents a massive thermodynamic driving force (5-6× more energy input than required for peptide bond) that pays for:

• Information transfer accuracy
• Directional ribosome movement
• Maintenance of reading frame
• High fidelity in amino acid selection

The high energy investment ensures accurate information transfer from nucleic acid to protein sequence.

• Peptide bond formation is catalyzed by peptidyl transferase, which is actually a ribozyme function of the 23S rRNA
• The α-amino group of the aminoacyl-tRNA in the A site acts as a nucleophile, attacking the ester linkage between the initiating fMet and its tRNA
• The N-formylmethionyl group transfers from the tRNA in the P site to the amino group of the second amino acid in the A site
• This reaction creates a dipeptidyl-tRNA in the A site, while the deacylated tRNAfMet remains in the P site
• After peptide bond formation, both tRNAs shift to a hybrid binding state:
  • The deacylated tRNA shifts so its 3' and 5' ends move to the E site
  • The dipeptidyl-tRNA shifts so its 3' and 5' ends move to the P site
  • The anticodons of both tRNAs remain in their original A and P sites
• This hybrid state prepares the ribosome for the subsequent translocation step

Note: The discovery that peptidyl transferase is a ribozyme, not a protein, provides important evidence for the RNA world hypothesis in evolution.

Stage 1: Amino acid activation

• Catalyzed by aminoacyl-tRNA synthetases
• Energy: ATP → AMP + PPi (equivalent to 2 ATP)
• Function: Forms aminoacyl-tRNAs
• Accuracy: Critical for translation fidelity; some enzymes have proofreading activity
• In bacteria: Initiator is N-formylmethionyl-tRNAfMet

Stage 2: Initiation

• Forms complex of ribosomal subunits, mRNA, initiator tRNA, and factors
• Energy: 1 GTP hydrolyzed to GDP + Pi
• Key components: 30S subunit, mRNA, GTP, fMet-tRNAfMet, initiation factors, 50S subunit

Stage 3: Elongation

• Adds amino acids sequentially to growing polypeptide chain
• Energy: 2 GTP per amino acid (1 for tRNA binding, 1 for translocation)
• Process: Aminoacyl-tRNA binding, peptidyl transfer, translocation
• Recycling: Deacylated tRNAs exit via E site

Stage 4: Termination

• Releases completed polypeptide with help of release factors
• Energy: At least 1 GTP hydrolyzed
• Total energy: ≥4 high-energy phosphate bonds per amino acid added

Stage 5: Folding and modification

• Protein folds into active 3D structure
• Energy: Indirect (chaperones may use ATP)
• Additional processing: Post-translational modifications
• Location: Begins in ribosome tunnel, continues in cytosol or destination compartment

The high energy investment (≥4 ATP/GTP equivalents per peptide bond) ensures translation accuracy.

Diphtheria toxin:

• Molecular weight: 58,330
• Mechanism: Catalyzes ADP-ribosylation of diphthamide (modified histidine) in eukaryotic elongation factor eEF2
• Effect: Inactivates eEF2, blocking translocation step of protein synthesis
• Enzymatic action: One toxin molecule can modify many eEF2 molecules
• Target: Affects all eukaryotic cells (not bacteria)

Ricin:

• Molecular weight: 29,895
• Source: Castor bean
• Mechanism: Depurinates a specific adenosine in 23S rRNA of 60S ribosomal subunit
• Effect: Inactivates ribosomes, preventing protein synthesis
• Enzymatic action: One ricin molecule can inactivate thousands of ribosomes per minute
• Target: Affects eukaryotic ribosomes

Potency factors:

1. Enzymatic (catalytic) rather than stoichiometric action
2. Target essential components of protein synthesis machinery
3. Affect all cellular proteins, not just a single pathway
4. Irreversible modification of targets
5. No repair mechanisms to quickly restore function

These toxins are lethal at extremely low doses due to amplification of their effects through catalytic action.

The pathway proceeds through multiple organelles in sequence:

1. Rough ER: Initial synthesis, folding, and modification (including N-linked glycosylation)
2. Transport vesicles: Carry proteins from ER to Golgi
3. Golgi complex: Further protein modification and sorting
   • Cis side: Receives proteins from ER
   • Medial region: Additional modifications
   • Trans side: Final sorting decisions occur here
4. Sorting vesicles: Direct proteins to specific destinations
5. Transport vesicles: Carry proteins to final destinations:
   • Lysosomes (via mannose-6-phosphate signaling)
   • Secretory granules (for regulated secretion)
   • Plasma membrane (for membrane proteins)
   • Extracellular space (for secreted proteins)

Note: This process is directional and compartmentalized, allowing precise control over protein trafficking and modification.

Regulatory Functions of the Ubiquitin-Proteasome System

1. Cell Cycle Regulation

   • Timed degradation of cyclins using "destruction box" sequences
   • Removal of cell cycle inhibitors to control G1→S→G2→M transitions

2. Immune System Function

   • Processing antigens for MHC class I presentation
   • Generation of antigenic peptides from intracellular proteins

3. Signal Transduction

   • Controls receptor abundance at cell membrane
   • Regulates transcription factor availability and activity
   • Modulates signaling pathway components for temporal control

4. Development and Stress Response

   • Orchestrates protein turnover during embryogenesis and tissue remodeling
   • Removes oxidatively damaged proteins during stress

Pathologies from Dysfunction

1. Cancer

   • Insufficient degradation of oncogene products
   • Excessive degradation of tumor suppressors (e.g., HPV E6-mediated p53 degradation)

2. Neurodegenerative Disorders

   • Protein aggregation in Alzheimer's (amyloid-β), Parkinson's (α-synuclein), and Huntington's (polyglutamine proteins)
   • Overwhelmed degradative capacity

3. Genetic and Inflammatory Conditions

   • Cystic fibrosis: excessive degradation of partially functional CFTR
   • Liddle's syndrome: impaired sodium channel degradation causing hypertension
   • Dysregulated NF-κB processing leading to inflammatory disorders

Molecular Mechanisms of Dysfunction

• Mutations in E3 ligases affecting substrate recognition
• Defects in deubiquitinating enzymes
• Impaired proteasome assembly or catalytic function
• Imbalance between protein misfolding and degradative capacity

Note: The specificity of this system relies on distinct E2-E3 enzyme combinations that target specific substrate proteins, creating a highly selective system for temporal and spatial protein regulation.

Bacterial Sec Protein Export Pathway:

1. Recognition & Prevention of Premature Folding:

   • Newly synthesized protein with signal sequence must remain unfolded
   • SecB chaperone binds to signal sequence and/or incompletely folded regions
   • SecB prevents premature folding that would inhibit export

2. Targeting to Membrane Complex:

   • SecB delivers unfolded protein to SecA at inner membrane
   • SecA functions as both receptor and ATP-dependent translocation motor
   • Protein transfers from SecB to SecA; SecB is released

3. Translocation Mechanism:

   • SecA associates with SecYEG translocation channel in membrane
   • SecA undergoes ATP-driven conformational changes that:
     • Insert SecA partially into membrane
     • Push ~20 amino acid segments through SecYEG channel
     • Withdraw from membrane upon ATP hydrolysis
   • Cycles of ATP binding/hydrolysis by SecA drive stepwise translocation
   • Signal sequence cleaved by signal peptidase during process

4. Completion of Export:

   • Entire protein passes through SecYEG complex into periplasm
   • Proton motive force (electrochemical potential) provides additional driving force
   • Protein completes folding after translocation

Key Features:

• Energy source: ATP hydrolysis by SecA (unlike eukaryotic GTP-dependent systems)
• Translocation occurs in ~20 amino acid increments
• Requires both SecA conformational cycling and membrane potential
• SecYEG forms the actual transmembrane channel