Lecture 10 (MCB 450)

What is the genetic code, and what is a codon?

The genetic code determines how mRNA nucleotide sequences specify amino acids. A codon is a sequence of three nucleotides that specifies an amino acid or a stop signal.

Key properties:

• Specific: Each codon has a defined meaning.

• Degenerate: Multiple codons can specify the same amino acid.

• Nearly universal: Most organisms use the same genetic code.

• Nonoverlapping and comma-less: Codons are read consecutively in groups of three.

What are the start and stop codons?

• AUG: The usual start codon; codes for methionine in eukaryotes and formylmethionine (fMet) at bacterial initiation.

• UAA, UAG, UGA: Stop codons that signal termination of translation.

How can point mutations, insertions, and deletions affect a protein?

• Silent mutation: Changes a codon but not the amino acid.

• Missense mutation: Changes one amino acid to another. The lecture uses sickle cell disease as an example.

• Nonsense mutation: Changes a codon into a stop codon, producing a shortened protein.

• Insertion or deletion: Adds or removes nucleotides. If the number is not a multiple of three, it causes a frameshift, changing downstream codons.

What is the wobble hypothesis?

• The wobble hypothesis explains how one tRNA can recognize multiple codons for the same amino acid.

• Pairing is flexible between the first base of the tRNA anticodon and the third base of the mRNA codon.

Why is wobble important?

• It allows cells to translate multiple synonymous codons without needing a different tRNA for every codon.

• The first two codon bases pair more strictly, while the third position allows flexible pairing.

What are the two main functions of aminoacyl-tRNA synthetases?

• Amino acid activation: Uses ATP to activate an amino acid, forming aminoacyl-AMP.

• tRNA charging: Transfers the correct amino acid to its matching, or cognate, tRNA.

Why are aminoacyl-tRNA synthetases important for translation accuracy?

They ensure that the correct amino acid is attached to the correct tRNA. Many also have editing activity to remove incorrectly charged amino acids. Errors can cause the wrong amino acid to be incorporated into a protein.

What are the A, P, and E sites of the ribosome?

• A site (aminoacyl): Accepts the incoming charged tRNA.

• P site (peptidyl): Holds the tRNA carrying the growing polypeptide chain.

• E site (exit): Where the uncharged tRNA exits.

What happens during polypeptide elongation?

The ribosome accepts the next charged tRNA, forms a peptide bond, and moves one codon along the mRNA. The growing chain is transferred to the incoming tRNA, and the empty tRNA eventually exits.

Translation reads the mRNA 5′ → 3′, while the protein grows from its N-terminus to its C-terminus.

How is the translation initiation site selected in bacteria?

• The Shine–Dalgarno sequence in the mRNA base-pairs with a complementary region of the 16S rRNA in the small ribosomal subunit.

• This positions the start codon correctly in the ribosome.

What roles do 16S rRNA, the initiator tRNA, and the start codon play?

• 16S rRNA: Helps recognize and position the mRNA.

• Shine–Dalgarno sequence: Helps align the start codon.

• AUG start codon: Establishes where translation begins.

• Initiator tRNA: Recognizes the start codon and brings the first amino acid, fMet in bacteria.

How does streptomycin inhibit protein synthesis?

Streptomycin binds the bacterial 30S ribosomal subunit, interfering with initiation and promoting errors in mRNA decoding.

How does tetracycline inhibit protein synthesis?

Tetracycline binds the bacterial 30S subunit and prevents incoming aminoacyl-tRNA from properly entering the A site.

How do chloramphenicol and erythromycin inhibit protein synthesis?

• Chloramphenicol: Inhibits peptidyl transferase activity on the bacterial 50S subunit, preventing peptide-bond formation.

• Erythromycin: Binds the bacterial 50S subunit and inhibits elongation, including ribosomal translocation

How do diphtheria toxin and ricin inhibit protein synthesis?

• Diphtheria toxin: ADP-ribosylates eukaryotic elongation factor 2 (eEF2), preventing ribosomal translocation.

• Ricin: Damages 28S rRNA in the eukaryotic 60S subunit, inactivating the ribosome.

Memory tip: Streptomycin and tetracycline target 30S; chloramphenicol and erythromycin target 50S. Diphtheria toxin targets an elongation factor, while ricin damages ribosomal RNA.

How does translation initiation differ between bacteria and eukaryotes?

• Bacteria: The Shine–Dalgarno sequence pairs with 16S rRNA to position the start codon.

• Eukaryotes: The small ribosomal subunit is recruited near the 5′ cap and scans along the mRNA to identify an AUG in an appropriate Kozak sequence context.

What initiator tRNA is used in bacteria versus eukaryotes?

• Bacteria: Initiator tRNA carries N-formylmethionine (fMet).

• Eukaryotes: Initiator tRNA carries methionine (Met).

Both systems use AUG as the usual start codon, but they recognize and position it differently.

How does the signal peptide–SRP pathway direct a protein to the rough ER?

1. Translation begins on a ribosome in the cytosol.

2. An ER signal sequence emerges from the growing protein.

3. The signal recognition particle (SRP) binds the signal sequence and ribosome.

4. The complex docks at the SRP receptor on the rough ER.

5. The ribosome engages a translocation channel, and translation continues as the protein enters the ER or is inserted into its membrane.

What is the difference between free and ER-bound ribosomes?

• Free ribosomes: Usually make proteins destined for the cytosol or, with appropriate targeting signals, the nucleus, mitochondria, or peroxisomes.

• ER-bound ribosomes: Make proteins entering the secretory pathway, including secreted proteins, many membrane proteins, and many lysosomal proteins.

Ribosomes are not fundamentally different; targeting signals determine where the translating ribosome goes.

What is phosphorylation, and which amino acids are commonly phosphorylated?

• Phosphorylation adds a phosphate group to a protein, often regulating its activity or signaling interactions.

• Common target residues are serine, threonine, and tyrosine.

What is acetylation, and which residues can be modified?

• Acetylation adds an acetyl group.

• It commonly occurs on the ε-amino group of lysine or the protein's N-terminal amino group.

• it can affect protein activity, stability, and interactions.

What is hydroxylation, and which amino acids are commonly modified?

• Hydroxylation adds a hydroxyl group. 

• Proline and lysine are important examples, particularly in collagen, where hydroxylation supports structural stability.

What is glycosylation, and which amino acids are commonly modified?

Glycosylation attaches carbohydrate groups to proteins.

• N-linked glycosylation: Commonly attaches sugars to asparagine.

• O-linked glycosylation: Commonly attaches sugars to serine or threonine.

It contributes to protein folding, stability, recognition, and trafficking.

What is ubiquitination?

• Ubiquitination attaches the small protein ubiquitin to a target protein, commonly through a lysine residue.

• Depending on the ubiquitin signal, it can affect protein degradation, localization, or activity.

How does ubiquitination target a protein for degradation?

Ubiquitin is attached through an enzyme cascade:

1. E1: Activates ubiquitin using ATP.

2. E2: Carries activated ubiquitin.

3. E3: Recognizes the target protein and helps transfer ubiquitin to it.

Repeated ubiquitin attachment can form a polyubiquitin chain that marks a protein for proteasomal degradation.

What does the proteasome do?

• The 26S proteasome recognizes appropriately tagged proteins, unfolds them, and degrades them into short peptides.

• The ubiquitin–proteasome pathway helps remove damaged, misfolded, or short-lived regulatory proteins.

Why is the ubiquitin–proteasome pathway important?

• it controls protein quality and abundance

• it removes proteins that should no longer function and helps regulate processes such as the cell cycle and signaling

What components are required for translation?

• Amino acids

• tRNAs

• Aminoacyl-tRNA synthetases

• mRNA

• Small and large ribosomal subunits

• Initiation, elongation, and termination factors

• Energy, including ATP and GTP

What are polyribosomes (polysomes)?

• Polyribosomes are groups of multiple ribosomes translating the same mRNA simultaneously.

• They allow many copies of a protein to be produced from one mRNA molecule.

How does the central dogma relate to translation?

• The central dogma describes the general flow of information: DNA → RNA → protein.

• Translation is the step in which the ribosome reads mRNA codons and uses tRNAs to assemble an amino-acid sequence into a protein.