Protein Synthesis in Eukaryotic Cells

Protein Synthesis in Eukaryotic Cells

• Learning Outcomes: Students will be able to describe the genetic code, the structure and function of tRNA, aminoacyl-synthetase, and ribosomes in eukaryotic protein translation, key events in eukaryotic protein synthesis, and mechanisms that regulate protein concentration.

Lecture Outline

• 1. The genetic code: A set of three nucleotides (codons) in mRNA that codes for specific amino acids. One amino acid can have more than one codon. This code is largely universal across organisms but with slight differences in mitochondria. Twenty different amino acids.
• 2. Key molecules in protein synthesis: These include tRNA (transfer RNA), aminoacyl-tRNA synthetases, and ribosomes.
• 3. Protein synthesis: This involves initiation, elongation, and termination steps.
• 4. Regulation of final protein concentration: Mechanisms for controlling the final concentration of a protein can affect mRNA expression, protein stability, and post-translational modifications.

Protein Expression Regulation

• Protein expression depends on regulation of mRNA expression and breakdown.
• Steps in this regulation: transcriptional control of mRNA, RNA processing control, mRNA transport and localization control, mRNA degradation control, translation control, protein activity control, and protein degradation control.

The Genetic Code

• Codons are sets of three nucleotides in mRNA that code for amino acids.
• There is a one-to-many relationship. More than one codon may specify the same amino acid.
• The code is largely universal but with some slight differences in the mitochondria.
• There are 20 amino acids in total.

Possible Reading Frames in mRNA Translation

• The reading frame in mRNA translation depends on the starting codon (5' to 3'), affecting the protein produced.
• There are three possible reading frames for mRNA translation.
• Correct reading frame is essential to produce a functional protein.
• Errors could result in amino acid misreading and production of nonfunctional proteins.
• The AUG codon is the start signal for translation.

The Role of tRNA in Translation

• tRNA (transfer RNA) carries amino acids to the ribosome.
• tRNA has a cloverleaf structure with an anticodon loop that binds to the complementary codon. Amino acid is covalently attached at the 3' end.
• The anticodon of tRNA pairs with the mRNA codon.
• Some tRNAs can tolerates a mismatch at 3rd position in the codon-anticodon binding.

Aminoacyl-tRNA Synthetases

• Aminoacyl-tRNA synthetases attach the correct amino acid to its corresponding tRNA molecule.
• Each amino acid has a specific synthetase.
• This process requires ATP and creates a "charged" tRNA.
• The synthetase ensures the correct amino acid is attached to the tRNA. This ensures the correct protein sequence is formed.

Charging of tRNA Molecules

• Charging means attaching an amino acid to tRNA.
• Aminoacyl-tRNA synthetases catalyze this process, building a bond between tRNA and the specific amino acid.
• A high-energy bond is created during this process.
• This reaction couples with ATP hydrolysis.
• Errors in charging can lead to incorrect amino acids being incorporated into the protein.

Translation on Ribosomes

• Ribosomes are the site of protein synthesis. Free or attached to the endoplasmic reticulum (ER). Large complexes made from ribosomal RNA (rRNA) and proteins.
• Small subunit: matches tRNA to mRNA codons.
• Large subunit: contains the catalytic site (for peptide bond formation).
• The ribosome has three binding sites binding sites for tRNAs (A, P, E sites).
• mRNA forms a template for protein synthesis.
• The ribosome moves along the mRNA guiding tRNA for the sequential addition of amino acids to the growing polypeptide chain.

Protein Synthesis: Initiation

• Requires various initiation factors and initiator tRNA (carrying methionine).
• The initiator tRNA binds to the P site on the small ribosomal subunit
• The small subunit binds to the 5' end of the mRNA (indicated by the cap).
• The ribosome scans along the mRNA searching for the start codon (AUG)
• After initiation the large subunit binds to the complex.
• Recognition of AUG codon leads to release of initiation factors

Protein Synthesis: Elongation

• New aminoacyl-tRNA enters the A site on the ribosome.
• Peptide bond formation between the carboxyl end of the growing polypeptide chain and the amino group of the incoming amino acid.
• Translocation of the large subunit.
• Translocation of the small subunit.
• The spent tRNA moves to the E site, then exits.

Protein Synthesis: Termination

• Stop codons (UAA, UAG, UGA) signal the termination of translation.
• Release factors bind to the stop codon.
• Peptidyl transferase activity is altered from catalyzing peptide bond formation to adding a water molecule.
• The polypeptide chain is released.
• The ribosome dissociates from the mRNA.

Proteins are Synthesized on Polyribosomes

• Multiple ribosomes can translate the same mRNA simultaneously (polyribosomes or polysomes).
• This increases the rate of protein synthesis for a given mRNA.

Post-translational Processing of Synthesized Proteins

• Polypeptides may require further modifications to become functional proteins.
• Folding into their correct 3D shape.
• Binding to cofactors or other proteins.
• Covalent modifications (e.g., phosphorylation).
• These modifications can regulate protein activity, location, and interactions.

Protein Concentration Regulation by Proteolysis

• Proteolysis is the breakdown of proteins.
• Proteases are enzymes that catalyze proteolysis.
• Proteasomes are large protein complexes that degrade proteins that are no longer needed by the cell.
• Damaged or misfolded proteins are often targeted for degradation by ubiquitin tagging.

Protein Expression Summary

• Protein expression depends on multiple regulation steps affecting mRNA (transcription through translation) and protein degradation processes including controls for protein folding and activity.