Protein Metabolism

Cracking the genetic code involved complementary experimental approaches:

1. Nirenberg and Matthaei's Homopolymer Experiment (1961): • Method: Synthetic RNA polymers (poly(U), poly(A), poly(C)) were added to cell-free protein synthesis systems • Results:

   • poly(U) → polyphenylalanine
   • poly(C) → polyproline
   • poly(A) → polylysine • Conclusion: UUU codes for Phe, CCC for Pro, AAA for Lys

2. Mixed Polymer Experiments: • Method: Random polymers with defined base ratios created using polynucleotide phosphorylase • Analysis: Correlation between base composition and amino acid incorporation frequencies • Example: A polymer with A:C ratio of 5:1 helped assign codons for multiple amino acids

3. Khorana's Repeating Sequence Method: • Method: Synthetic polymers with defined repeating sequences (e.g., ACACAC...) • Analysis: Produced polypeptides with repeating amino acids • Example: (AC)n produced alternating threonine and histidine, showing ACA codes for Thr and CAC for His

4. Trinucleotide Binding Assay (Nirenberg and Leder, 1964): • Method: Specific aminoacyl-tRNAs bound to ribosomes in presence of synthetic trinucleotides • Results: Direct association between specific codons and amino acids

Scientific significance: • Demonstrated the triplet nature of the code • Proved codons are universal (with minor exceptions) • Established the nonoverlapping, degenerate nature of the code • Represented one of biochemistry's greatest achievements, completed in just five years (1961-1966) • Fundamental to understanding how genetic information is translated into proteins

Key evidence from ribosome structure supporting the RNA world hypothesis:

1. The peptidyl transferase center (the active site for peptide bond formation) consists entirely of rRNA, with no protein within 18Å
2. The ribosome is fundamentally a ribozyme - RNA catalyzes the critical peptide bond formation
3. Ribosomal proteins are secondary elements, primarily decorating the surface of the RNA core
4. The peptidyl transferase center sequence is highly conserved across all species
5. Artificial ribozymes that promote peptide synthesis contain the conserved sequence (5')AUAACAGG(3') found at the peptidyl transferase site

This suggests protein synthesis was originally catalyzed by RNA molecules, supporting the theory that RNA predated proteins in early evolution.

Two-step mechanism:

Step 1: Amino acid activation

• Amino acid + ATP → aminoacyl-AMP + PPi
• Carboxyl group of amino acid reacts with α-phosphoryl of ATP
• Forms enzyme-bound aminoacyl adenylate intermediate
• Release of pyrophosphate

Step 2: Transfer to tRNA

• For Class I synthetases: aminoacyl group transfers to 2'-OH of tRNA, then to 3'-OH
• For Class II synthetases: aminoacyl group transfers directly to 3'-OH of tRNA
• Aminoacyl-AMP + tRNA → aminoacyl-tRNA + AMP

Energetics:

• PPi hydrolysis to 2Pi by pyrophosphatase drives first reaction forward
• Overall reaction consumes two high-energy phosphate bonds
• Final aminoacyl-tRNA ester linkage has high negative free energy of hydrolysis (ΔG° = -29 kJ/mol)
• Reaction is essentially irreversible

Net reaction: Amino acid + tRNA + ATP → aminoacyl-tRNA + AMP + 2Pi

The Dintzis experiment proved that polypeptides grow by sequential addition of amino acids to the carboxyl-terminal end.

Key points: • When radioactive leucine was added to actively synthesizing hemoglobin, the label appeared first only at the carboxyl end of completed chains • As time progressed (4 min → 7 min → 16 min → 60 min), the radioactive region extended progressively toward the amino terminus • This pattern conclusively demonstrated that protein synthesis begins at the amino terminus and proceeds toward the carboxyl terminus • This established the fundamental directionality of ribosomal protein synthesis in all organisms

This principle is essential for understanding how ribosomes translate mRNA in the 5'→3' direction to create proteins with amino→carboxyl directionality.

• 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.

Bacterial translation termination occurs in three sequential steps:

1. Recognition: When a termination codon (UAG, UAA, or UGA) enters the A site, a release factor (RF-1 or RF-2) binds to it

   • RF-1 recognizes UAG and UAA
   • RF-2 recognizes UGA and UAA

2. Hydrolysis: The release factor induces peptidyl transferase to transfer the completed polypeptide to a water molecule instead of another amino acid

   • This hydrolyzes the ester bond between the nascent polypeptide and the tRNA in the P site
   • The completed polypeptide is released with a free carboxyl group (COO-)

3. Dissociation: The ribosomal complex disassembles

   • The mRNA, deacylated tRNA, and release factor leave the ribosome
   • The 70S ribosome dissociates into its 30S and 50S subunits

RF-3 is thought to assist in releasing the ribosomal subunit, though its specific function is not firmly established.

Farnesylation is a post-translational modification in which:

• A farnesyl group (derived from farnesyl pyrophosphate) is covalently attached to a cysteine residue of a protein
• A thioether linkage (S-C bond) is formed between the sulfhydryl group of cysteine and the farnesyl group
• Pyrophosphate (PPi) is released as a byproduct
• The modification creates a lipid anchor that helps target proteins to membranes

Example: Ras proteins (products of ras proto-oncogenes) require farnesylation for proper membrane localization and signaling function.

Inhibition of farnesylation is being explored for cancer treatment because:

• Ras proteins, which are products of ras oncogenes, require farnesylation to function
• Approximately 30% of human cancers involve activating mutations in ras genes
• Without farnesylation, Ras proteins cannot anchor to cell membranes
• Membrane localization is essential for Ras to activate downstream signaling pathways involved in cell proliferation
• Blocking farnesylation can inhibit the transforming (carcinogenic) activity of mutant Ras
• Farnesyltransferase inhibitors potentially disrupt oncogenic signaling with minimal effects on normal cells

Note: The therapeutic challenge is achieving specificity, as many proteins beyond Ras are also farnesylated.

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.

SRP Composition:

• Ribonucleoprotein complex containing one RNA molecule and six protein subunits
• Recognizes hydrophobic signal sequences on emerging polypeptides

Sequence of Events:

1. As polypeptide with signal sequence emerges from ribosome, SRP binds to the signal sequence
2. SRP simultaneously binds to the ribosome, causing translation elongation to pause (arrest)
3. The entire SRP-ribosome-nascent chain complex moves to the ER membrane
4. SRP binds to the SRP receptor (SR) in the ER membrane
5. GTP binding occurs to both SRP and SR
6. The ribosome-nascent chain complex is transferred to the translocon (Sec61 complex)
7. GTP hydrolysis occurs, releasing SRP and SR from each other
8. SRP returns to the cytosol for another round of targeting
9. Translation resumes, with the growing polypeptide moving directly into the ER lumen
10. Signal peptidase cleaves the signal sequence inside the ER lumen

This co-translational process ensures that secretory and membrane proteins are properly inserted into the ER before folding occurs.