Protein Metabolism

Components required for amino acid activation:

• 20 amino acids (the building blocks)
• 20 aminoacyl-tRNA synthetases (enzymes specific to each amino acid)
• 32 or more tRNAs (adaptor molecules)
• ATP (energy source)
• Mg²⁺ (cofactor)

This stage occurs in the cytosol, not on the ribosome, and involves "charging" tRNAs by covalently attaching the correct amino acid to its corresponding tRNA, creating aminoacyl-tRNAs that will be used in subsequent stages.

5S rRNA is structurally distinct from 16S rRNA in several ways:

• Much smaller size (approximately 120 nucleotides vs. 1,542 for 16S)
• Simpler secondary structure with fewer helical regions and loops
• Contains a characteristic Y-shaped core with three stem-loops
• Located in the large (50S) ribosomal subunit rather than the small (30S) subunit

Functional significance:

• Serves as a structural scaffold within the 50S subunit
• Forms part of the central protuberance of the ribosome
• Helps coordinate interactions between the large and small subunits
• Assists in positioning tRNAs during translation
• Contributes to ribosomal stability without direct involvement in peptide bond formation

The structural simplicity of 5S rRNA reflects its more limited role in translation compared to the catalytically crucial 16S and 23S rRNAs.

Modified nucleotides in tRNA serve several critical functions:

1. Structural stabilization - Modifications like dihydrouridine (D) alter RNA's backbone flexibility
2. Base-pairing precision - Pseudouridine (Ψ) enhances base-stacking and increases hydrogen bonding
3. Codon recognition - Modified bases in the anticodon loop (like inosine) expand codon recognition capabilities
4. Protection from degradation - Methylated bases prevent nuclease attack
5. Discrimination between tRNAs - Creating unique structural signatures for aminoacyl-tRNA synthetase recognition

For example, in yeast tRNAᴬˡᵃ, modifications include pseudouridine, inosine, ribothymidine, dihydrouridine, methylinosine, methylguanosine, and dimethylguanosine, each serving specific roles in maintaining proper tRNA function.

Discrimination between initiating and internal AUG codons depends on:

1. Specialized initiator tRNA (tRNAfMet):

   • Structurally distinct from elongator tRNAMet
   • Carries N-formylmethionine (preventing use at internal positions)
   • Binds directly to the P site (unlike all other aminoacyl-tRNAs that enter A site)

2. Contextual positioning:

   • Initiating AUG is positioned near Shine-Dalgarno sequence
   • The Shine-Dalgarno sequence base-pairs with 16S rRNA
   • This interaction precisely positions the AUG in the P site

3. Ribosomal recognition:

   • Specific interactions between ribosomal P site and fMet-tRNAfMet
   • The formyl group prevents binding at internal codons
   • Internal AUG codons are decoded by Met-tRNAMet entering through the A site

This system ensures initiator AUG is distinguished from internal AUG solely by context and specialized initiator tRNA

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.

• Amino acid arm (residues 1-7, 72):

  • Contains the CCA sequence at the 3' end (position 72)
  • The terminal adenosine's ribose is the attachment point for amino acids
  • Forms one branch of the L-shape

• TΨC arm (residues 54-64):

  • Contains modified nucleotides (ribothymidine and pseudouridine)
  • Interacts with the large ribosomal subunit during translation
  • Contributes to overall tRNA stability through tertiary interactions

• D arm (residues 10-25):

  • Named for dihydrouridine modifications
  • Creates the bend in the L-shape
  • Position 20 is involved in tertiary structure stabilization

• Anticodon arm (residues 32-44):

  • Anticodon loop exposed at positions 32-38
  • Position 34 (first anticodon position) often contains modified bases to allow wobble pairing
  • Forms the opposite branch of the L-shape from the amino acid arm

The precise spatial arrangement of these arms ensures both the structural integrity of tRNA and the exact positioning required for its adaptor function in translation.

The "second genetic code" refers to the specific recognition mechanism between aminoacyl-tRNA synthetases and their cognate tRNAs:

• It determines which amino acid gets attached to which tRNA, ensuring translation fidelity
• Recognition involves specific nucleotide positions in tRNAs that serve as identity elements:
  • Conserved nucleotides (common to all tRNAs) cannot be used for discrimination
  • Some positions are recognition points for only one specific synthetase
  • Other positions may be recognized by multiple synthetases
• Both sequence and structural features contribute to recognition
• This recognition system is critical for maintaining the accuracy of protein synthesis

Example: In alanine tRNAs across all organisms, a single G-U base pair in the amino acid arm serves as the primary recognition element for the Ala-tRNA synthetase.

• Reaction positioning:
  • Places ATP adjacent to the 3' terminal adenosine of tRNA
  • Creates optimal geometry for the two-step aminoacylation reaction
  • Ensures the amino acid can be transferred to the correct position on tRNA
• Mechanistic requirements:
  • Facilitates formation of the aminoacyl-adenylate intermediate
  • Positions the activated amino acid for transfer to the tRNA
  • The proximity minimizes side reactions with water or other molecules
• Energy coupling:
  • ATP hydrolysis provides energy to form the high-energy ester bond
  • Strategic positioning ensures energy is efficiently transferred to bond formation
  • Prevents wasteful ATP hydrolysis when tRNA is not properly bound

Example: In glutaminyl-tRNA synthetase, the ATP binding pocket positions the α-phosphate group of ATP precisely where it can react with glutamine, forming glutaminyl-AMP that then transfers the glutaminyl group to the properly positioned 3' end of tRNAGln.

• The tRNAAla system demonstrates extreme recognition economy (single G=U base pair) compared to most tRNA-synthetase systems that utilize:
  • Multiple contact points across different tRNA arms
  • Anticodon recognition
  • Complex tertiary structure elements
• This illustrates that biological specificity can be achieved through remarkably minimal determinants
• It demonstrates that:
  • Not all biomolecular recognition requires extensive interaction surfaces
  • Single structural features can confer exquisite specificity
  • Evolution can select for simplicity when it achieves the needed function

Example: While the glutamine tRNA synthetase recognizes multiple elements in both the anticodon and acceptor stem regions, the alanine system achieves equal specificity through a single wobble base pair

Pathogen Exploitation of Receptor-Mediated Endocytosis:

1. Viral Entry Mechanisms:

   • Influenza virus: Binds to sialic acid receptors, enters via clathrin-coated pits
   • Utilizes endosomal acidification to trigger conformational changes in viral proteins
   • Hemagglutinin-mediated fusion with endosomal membrane releases viral genome

2. Bacterial Toxin Entry:

   • Diphtheria toxin: Binds to specific cell surface receptor
   • Enters via clathrin-dependent endocytosis
   • Low pH in endosomes triggers conformational change
   • A domain of toxin inserts into membrane, allowing B fragment to enter cytosol
   • Inhibits protein synthesis by ADP-ribosylation of elongation factor 2

3. Cholera Toxin Entry:

   • Binds to GM1 ganglioside receptors via B subunit
   • Enters cells through endocytosis
   • A subunit activates adenylate cyclase, increasing cAMP levels
   • Results in excessive fluid secretion and diarrhea

These pathogens have evolved to exploit normal cellular uptake processes, targeting specific receptors and utilizing vesicular trafficking and pH changes to optimize their cellular invasion.