Comprehensive Study Guide on Translation, Protein Synthesis, and Genetic Expression

Final Exam Information

• Exam Size: The final exam will consist of approximately $50$ questions.
• Duration: Students will have a total of $2$ hours to complete the exam.
• Materials: A codon table will be provided for the exam. It may be included as a file accompanying specific test questions or brought in by the student to start the test. There will be at least one question on the exam requiring the translation of an mRNA sequence.

Homework Review: Mutation and Translation Analysis

• Starting Translation: Translation always begins at the start codon sequence $AUG$.
      - In the provided homework sequence, the $AUG$ sequence starts at approximately the fifth letter.
      - $AUG$ codes for the amino acid Methionine.
• Codon Structure: Codons are three-letter sequences of nucleotides.
• Stop Codons: There are three possible stop codons that signal the end of translation: $UAA$, $UAG$, and $UGA$.
      - In the original homework sequence, the stop codon used is $UGA$.
      - Stop codons do not code for an amino acid; the last amino acid in the chain is the one immediately preceding the stop codon.
• Translation of Original Sequence:
      - $AUG \rightarrow Methionine$
      - $GUA \rightarrow Valine$
      - $AUA \rightarrow Isoleucine$
      - $ACA \rightarrow Threonine$
      - $CAU \rightarrow Histidine$
• The Mutated Strand (Insertion Mutation):
      - The mutation involves the insertion of an extra Uracil ($U$) nucleotide.
      - This results in a sequence approximately five amino acids long before reaching a stop codon ($UGA$) sooner than in the original sequence.
      - Mutated Translation Provided: Methionine, Valine, Acinine, Acinine, and Threonine.
• Impact of Mutation:
      - The protein is significantly shorter and contains different amino acids.
      - This changes the shape of the final protein, affecting its overall structure.
      - Such mutations more than likely render the protein unusable by the cell.

Specific Types of Insertion Mutations

• Frame Shift Mutation (Out of Frame Insertion):
      - Occurs when one or two letters are inserted into the sequence.
      - This shifts the reading frame for all subsequent codons, leading to a completely different amino acid sequence.
      - The homework example is a frame shift mutation because an odd number (one) of nucleotides was inserted.
• In-Frame Insertion Mutation:
      - Occurs when insertions happen in groups of three (or factors of three).
      - This results in the addition of an extra amino acid to the protein without shifting the reading frame of the rest of the sequence (unless a stop codon is created).
• Structural Consequences: Mutations often result in proteins being the wrong size, the wrong shape, and malformed.

The Process of Translation: Initiation

• Small Ribosomal Subunit: Also known as the $40s$ subunit in eukaryotes.
• Prokaryotic Initiation: The small subunit binds to the Shine Dalgarno sequence at the beginning of the mRNA. This positions the subunit correctly over the $AUG$ start codon.
• Eukaryotic Initiation: The mRNA has a $5'$ cap. The small ribosomal subunit attaches to this cap and scans along the strand until it identifies the first $AUG$ sequence.
• tRNA Binding: The first tRNA, typically carrying Methionine, base pairs with the $AUG$ start codon at the P site. All proteins begin with Methionine, though it may be removed later during editing.
• Full Assembly: The large ribosomal subunit ($60s$ in eukaryotes) joins to complete the ribosome assembly.
• A Site Status: Upon completion of initiation, the A site is empty and ready to accept the next tRNA-amino acyl combination.

The Process of Translation: Elongation

• Step 1: Decoding: A charged tRNA enters the A site. The anticodon on the tRNA base pairs with the mRNA codon. If the match is correct, the amino acid is accepted; if incorrect, it is rejected and another tRNA tries to bind.
• Step 2: Peptide Bond Formation: A peptide bond forms between the amino acid in the A site and the growing polypeptide chain in the P site. This reaction is catalyzed by the large ribosomal subunit ($60s$ subunit).
• Step 3: Translocation: The ribosome slides down the mRNA in the $5' \rightarrow 3'$ direction.
      - The tRNA carrying the chain moves from the A site to the P site.
      - The empty tRNA from the P site moves to the E site (Exit site) and is ejected from the ribosome.
      - The A site becomes empty, exposing a new codon to accept the next tRNA.

The Process of Translation: Termination

• Recognition: Termination begins when a stop codon ($UAA$, $UAG$, or $UGA$) enters the A site.
• Release Factor: Because there are no tRNA matches for stop codons, no amino acid attaches. Instead, a release factor protein enters the A site.
• Hydrolysis: The release factor triggers the hydrolysis of the bond between the polypeptide chain and the tRNA in the P site.
• Dissociation: The completed polypeptide is released. Subsequently, the mRNA, tRNA, and ribosomal subunits all dissociate. The subunits are then free to reassemble on a new mRNA for another round of translation.

Protein Folding and Quality Control

• Post-Translational Folding: After release, the protein is a linear chain of amino acids that must fold into a three-dimensional shape to be functional.
• Driving Forces: Folding is driven by:
      - Hydrophobic Reactions: Hydrophobic amino acids cluster on the interior of the protein to stay away from the water in the cytoplasm.
      - Hydrogen Bonds and Ionic Reactions: These stabilize the three-dimensional structure.
• Molecular Chaperones: Specialized proteins that assist in the folding process.
      - HSP 70: A major chaperone that binds to newly synthesized chains, guides them to the endoplasmic reticulum, and prevents incorrect folding or sticking to other proteins.
• Protein Degradation: If a protein misfolds or is malformed due to mutation, it is tagged with Ubiquitin and sent to a Proteasome (the digestive vacuole of the cell) to be dissolved.
• Functionality: For proteins like enzymes, the specific $3D$ shape is vital for holding molecules in the correct orientation to facilitate reactions. Incorrect folding results in a nonfunctional protein.

Selective Antibiotic Targeting of Protein Synthesis

Antibiotics can target prokaryotic ribosomes without affecting eukaryotic cells due to structural differences.

• Tetracyclines (e.g., Doxycycline, Tetracycline):
      - Mechanism: Block the A site of the $30s$ ribosomal subunit.
      - Result: tRNA cannot enter, halting translation.
      - Used for: Lyme disease, skin infections, Chlamydia, Rocky Mountain spotted fever, and other tick-borne diseases.
• Macrolides (e.g., Azithromycin/Z-Pak, Erythromycin):
      - Mechanism: Bind to the $50s$ subunit and block the polypeptide exit tunnel.
      - Result: Prevents the chain from elongating.
      - Used for: Respiratory infections, strep throat, and certain STIs.
• Aminoglycosides (e.g., Streptomycin, Gentamicin):
      - Mechanism: Block the small $30s$ subunit in bacteria.
      - Result: Causes codons to be misread; wrong amino acids are incorporated, creating nonfunctional proteins.
      - Used for: Serious infections, notably Tuberculosis (TB).

Comparison of Prokaryotic and Eukaryotic Translation

Shared Features

• mRNA Template: Read in the $5' \rightarrow 3'$ direction.
• Adapters: tRNA matches codons with amino acids.
• Ribosome Sites: Both use $A, P,$ and $E$ sites.
• Start Codon: Universally $AUG$ for Methionine.
• Stop Codons: Universally $UAA$, $UAG$, and $UGA$ utilizing release factors.
• Requirement: Proteins must fold after synthesis.

Key Differences

• Ribosome Size:
      - Prokaryotes: $70s$ ribosome (composed of $50s$ and $30s$ subunits).
      - Eukaryotes: $80s$ ribosome (composed of $60s$ and $40s$ subunits).
• Ribosome Recruitment:
      - Prokaryotes: Small subunit looks for the Shine Dalgarno sequence.
      - Eukaryotes: Small subunit looks for the $5'$ cap.
• First Amino Acid:
      - Both use Methionine, but prokaryotes utilize a slightly varied shape of the molecule.
• Location and Coupling:
      - Prokaryotes: Occurs in the cytoplasm; transcription and translation are coupled (happen simultaneously) because there is no nucleus.
      - Eukaryotes: Transcription occurs in the nucleus; mRNA must undergo processing and nuclear export. Translation occurs only in the cytoplasm.
• Antibiotic Sensitivity: Prokaryotic $70s$ ribosomes are sensitive to specific antibiotics that do not affect eukaryotic $80s$ ribosomes.

Summary of the Central Dogma and Gene Expression

• The Dogma: DNA $\rightarrow$ mRNA $\rightarrow$ Protein. (Rare exception: RNA $\rightarrow$ DNA in viruses like HIV).
• The Genetic Code:
      - Three-letter alphabet.
      - $64$ possible codons: $61$ for amino acids and $3$ stop codons.
      - Universal: Used by every organism on Earth.
      - Degenerate/Wobble: Multiple codons can code for the same amino acid. The first two letters are most critical; the third letter is the "wobble" location where changes may not alter the amino acid.
• Prokaryotic Summary:
      - Uses RNA polymerase and Sigma factor.
      - Promoter located at $-10$ to $-35$.
      - Termination via Rho factor or hairpins.
• Eukaryotic Summary:
      - Uses RNA polymerase II and transcription factors.
      - Promoter is the TATA box; guided by enhancers.
      - Pre-mRNA Processing: Includes $5'$ cap, splicing (removing introns, joining exons), and $3'$ poly-A tail.
      - Termination Site: $AAUAA$ sequence.
      - Splicing Rules: Follows the $GUAG$ rule via the spliceosome.
      - Alternative Splicing: $90\% - 95\%$ of genes undergo this, allowing $20,000$ genes to encode over $100,000$ different proteins.

Questions & Discussion

• Audience Member: Lyman students, are you able to see and hear me? Is it working on your end?
• Response: (No verbal response captured, technical connection issues noted).
• Audience Member: Do you guys want the or at least for for the Zoom, students, do you guys thinking that you want to, do independent study with the blue kit review as a homework, or do you guys wanna meet back for, for a class on Thursday, and I can do one of the the games that we did last time?
• Response: Is it possible to have both? To do both?
• Instructor: Yeah. That's fine. I'll go ahead and get that open. You should it should be an announcement on D2L here just a little bit after, after class… on Thursday, if you wanna join in for the the review games, I'll be here.
• Audience Member: I just wanna let you know that I'm gonna be leaving.
• Instructor: Yeah. Okay. Yeah. Thanks for thank you for letting me know.