Biological Translation and Ribosome Function

Administrative Updates and Exam Logistics

• Life Points Status: The live points for all students have been officially recorded. Students are encouraged to provide feedback through evaluations if they have time, though these will not change the point status.

• Final Exam Logistics:     * Date: Tuesday.     * Location: The exam will not be held at McBride; students must check their records for the specific finals room to avoid the common mistake of arriving at the regular classroom.     * Materials: A calculator is permitted for the exam.     * Time Duration: There is a full two-hour block allotted for the exam. This is designed to minimize time pressure, providing more time per question than midterms.

Biological vs. Synthetic Protein Synthesis

• The Amodator: A specialized machine used in organic labs for flow chemistry to synthesize short proteins without the assistance of biological cells.

• Comparison of Efficiency:     * Synthetic synthesis: The Amodator forms a peptide bond at a rate of approximately one every two minutes.     * Biological synthesis: Cells perform the same translation in a matter of seconds, making the process orders of magnitude faster than current human engineering.

• Evolutionary Engineering: The ribosome is a remarkable example of natural engineering, achieving efficiencies that synthetic systems cannot yet match.

Fundamental Concepts of Translation

• Definition: Translation is the successive formation of peptide bonds between separate amino acids to create long, functional polypeptides that fold into proteins.

• Directionality: Translation occurs in an ordered way from the amino ($N$) terminus to the carboxy ($C$) terminus. This parallels the $5'$ to $3'$ construction direction of nucleic acids.

• General Mechanism:     * tRNAs charged with amino acids bring the next unit to the carboxy terminus.     * The existing peptide chain is passed from the "old" tRNA to the "new" incoming tRNA.     * The de-acylated tRNA then exits the ribosome.

Ribosomal Structure and Active Sites

• Composition: Ribosomes are large macromolecules consisting of significant protein contributions and ribosomal RNA (rRNA). The RNA component performs the actual catalytic work (the peptidyl transferase activity).

• Subunits (Bacterial/Prokaryotic):     * $30S$ Small Subunit: Interacts directly with mRNA and identifies the start site.     * $50S$ Large Subunit: Interacts with the tips of tRNAs (where amino acids are attached) and contains the exit tunnel for the peptide.     * $70S$ Ribosome: The complete, functional complex.

• Functional Sites (APE Order):     * A (Aminoacyl) Site: Where the incoming charged (aminoacyl) tRNA arrives.     * P (Peptidyl) Site: Holds the tRNA attached to the growing polypeptide chain. It includes the exit tunnel for the nascent peptide.     * E (Exit) Site: Where de-acylated tRNAs move before being released from the ribosome.

The Five Stages of Translation

1. Activation: The charging of tRNAs with amino acids (covered in previous lectures).

2. Initiation: Finding the start site and laying down the first amino acid. Requires Initiation Factors (IFs).

3. Elongation: The physical movement of the ribosome relative to mRNA and the continuous addition of amino acids. Requires Elongation Factors (EFs).

4. Termination: Reaching a stop codon and cutting the final linkage between the tRNA and the peptide. Requires Release Factors (RFs).

5. Folding and Sorting: Post-translational processes involving signals for protein destination.

Bacterial Initiation Mechanics

• Initiator tRNA ($fMet$):     * The first amino acid added is always methionine, specifically $N$-formylmethionine ($fMet$) in bacteria.     * Structure: The formal group is attached to the amino group of methionine.     * Function: Unlike standard methionine, $fMet$ cannot be a substrate for further N-terminal addition because the amino group is "blocked." This marks the specific beginning of the protein.     * Synthesis: A transformylase moves a formal group from tetrahydrofolate ($THF$) to methionine after it has been charged onto the special tRNA ($tRNA_f$).

• Initiation Factors:     * $IF1$ and $IF3$: Bind to the $30S$ subunit to block the premature assembly of the full $70S$ ribosome.     * $IF2$: A GTPase protein that escorts the $fMet-tRNA_f$ specifically to the P site.

• Shine-Dalgarno Sequence:     * A purine-rich region in the mRNA (upstream of the start codon) that base-pairs with the $16S$ rRNA in the $30S$ subunit.     * This interaction precisely positions the start codon ($AUG$ or sometimes $GUG$) into the P site of the ribosome.     * Polycystronic mRNAs: In bacteria, a single mRNA can have multiple Shine-Dalgarno sequences to initiate the translation of multiple distinct proteins (e.g., the lac operon).

• Formation of the Complex: Once $IF2$ brings the initiator tRNA to the P site, the $30S$ pre-initiation complex is formed. $IF1$ and $IF3$ depart, the $50S$ subunit joins, $IF2$ hydrolyzes its GTP and departs, resulting in the $70S$ initiation complex.

Elongation Mechanics in Bacteria

• $EF-Tu$ (Elongation Factor Thermo Unstable):     * A highly abundant GTPase that escorts every aminoacyl tRNA (except the initiator) to the A site.     * Protection: It binds the tRNA tip, protecting the fragile ester linkage between the tRNA and amino acid from spontaneous hydrolysis by water.     * Proofreading: GDP hydrolysis only occurs if there is a correct match between the codon and anticodon.

• $EF-Ts$ (Elongation Factor Thermo Stable): Functions as a Guanine Nucleotide Exchange Factor (GEF) to reset $EF-Tu$ by replacing GDP with GTP.

• Peptidyl Transferase Reaction:     * Catalyzed by the $23S$ rRNA of the large subunit.     * The amino group of the tRNA in the A site performs a nucleophilic attack on the ester linkage of the peptidyl tRNA in the P site.     * Result: The entire peptide chain is transferred to the tRNA in the A site.

• Translocation and $EF-G$:     * To continue, the ribosome must move three nucleotides downstream.     * $EF-G$ (GTPase): Also known as the "translocase," it acts like a paddle or lollipop, providing a physical shove to move the tRNAs from A to P and P to E. This requires GTP hydrolysis.

Termination and Release

• Stop Codons: $UGA$, $UAA$, and $UAG$. These do not bind to any tRNAs.

• Release Factors (RFs):     * $RF1$ and $RF2$: Proteins that mimic the structure of tRNA and recognize stop codons in the A site.     * Mechanism: They allow a water molecule to enter the peptidyl transferase center, catalyzing the hydrolysis of the ester bond and releasing the protein.

• $RF3$: A GTPase that assists in the release of $RF1$ or $RF2$ after the protein has departed.

• Recycling: $EF-G$ and other factors help blow the ribosome complex apart so the subunits can be reused.

Eukaryotic Translation Differences

• Ribosome Size: Eukaryotic ribosomes ($80S$) are larger and more complex than bacterial ribosomes ($70S$).

• Initiation and Scanning:     * Does not use the Shine-Dalgarno sequence.     * $eIF4E$: Binds the $5'$ cap of the mRNA.     * Scanning: The small subunit, pre-bound with initiator methionine tRNA ($Met-tRNA_i$), starts at the $5'$ end and marches nucleotide-by-nucleotide until it finds the first $AUG$. This is an ATP-dependent process requiring helicase activity.

• Circularity (Uroboros):     * Eukaryotic mRNAs are circularized through interactions between the $5'$ cap (bound by $eIF4E$), a bridge protein ($eIF4G$), and the Poly-A Binding Protein ($PABP$) at the $3'$ tail.     * Purpose: This may act as a quality control mechanism or increase efficiency by keeping terminated ribosomes near the start site.

Clinical Applications: Antibiotics

• Selective Toxicity: Differences between prokaryotic and eukaryotic ribosomes allow antibiotics to target bacterial translation without harming the host.

• Examples:     * Streptomycin: High affinity for the bacterial $30S$ subunit; blocks the binding of $fMet-tRNA_f$. It targets the initiation phase.     * Puromycin: Mimics an aminoacyl-tRNA. It enters the A site and attaches to the growing peptide chain, causing premature chain termination and the release of abortive, short peptides. It targets the elongation phase.

Protein Sorting: The Secretory Pathway

• Signal Sequence: Targeted proteins (for secretion or the ER) have a sequence of approximately $10$ amino acids, usually alpha-helical, at the $N$-terminus.

• Signal Recognition Particle (SRP): This ribonucleoprotein binds to the signal sequence as it emerges from the ribosome and halts translation.

• ER Targeting: The SRP-ribosome complex binds to the SRP receptor on the Endoplasmic Reticulum (ER).

• Translocon: Both SRP and its receptor are GTPases. Upon hydrolysis, the translocon opens, translation resumes, and the protein is extruded (squirted) directly into the ER lumen. This process is often co-translational.

Regulation of Translation: The Iron Response System

• Proteins Involved:     * Ferritin: Stores iron within cells in a hollow 24-subunit ball; sequesters thousands of iron atoms.     * Transferrin Receptor: Responsible for the import of iron from the blood into the cell.

• Cis-Acting Element: Iron Response Element ($IRE$), a stem-loop structure in the mRNA.

• Trans-Acting Factor: Iron Response Element Binding Protein ($IREBP$). It binds iron when concentrations are high; when iron is low, it binds the $IRE$ on mRNA.

• Ferritin Regulation (5' UTR):     * Low Iron: $IREBP$ binds $IRE$ in the $5'$ UTR, physically blocking the ribosome. No translation.     * High Iron: $IREBP$ binds iron and leaves the mRNA. Translation proceeds to make ferritin for iron storage.

• Transferrin Receptor Regulation (3' UTR):     * Low Iron: $IREBP$ binds $IRE$ in the $3'$ UTR, masking instability signals and stabilizing the mRNA. This increases translation to import more iron.     * High Iron: $IREBP$ binds iron and leaves the mRNA. The unprotected mRNA is quickly degraded, decreasing iron import.