Cell S&F Lecture 32

Compare the overall size (S value) of ribosomes in Prokaryotes (Bacteria) vs. Eukaryotes.

Prokaryotic (Bacterial): 70S

Eukaryotic: 80S

Break down the 70S and 80S ribosomes into their two main subunits.

• 70S (Bacteria): 50S (Large) + 30S (Small)

• 80S (Eukaryotes): 60S (Large) + 40S (Small)

Where are eukaryotic ribosomes located, and what determines their two "states"?

• Free form: Suspended in the cytosol (usually making proteins for use inside the cell).

• Bound form: Attached to the ER membrane (making proteins for secretion or membranes).

mRNA binding site for ribosomes and polypeptides

• 5’ to 3’ for RNA

• N terminus to C terminus for polypeptides

A binding site

on the ribosome where mRNA attaches to initiate translation.

• newly Arrived

P binding site

on the ribosome where the tRNA carrying the growing polypeptide chain is located, facilitating peptide bond formation.

• tRNA Peptide chain

E binding site

on the ribosome where the uncharged tRNA exits after delivering its amino acid.

• tRNA Exit

Adaptor molecules

molecules that mediate the transfer of specific amino acids to the growing polypeptide chain during translation.

• help amino acids recognize the nucleotide sequences on the mRNA

tRNA

binds amino acids (1 ea.) AND recognize mRNA codons (1 or more)

Anti-codon

a three-nucleotide sequence on tRNA that pairs with the corresponding codon on mRNA during protein synthesis.

how many different tRNAs are found in the cells

Fewer than 61 (but more than 20)

The Wobble Hypothesis

proposes that the third position of a tRNA anticodon can pair with more than one nucleotide in the corresponding mRNA codon, allowing flexibility in codon recognition.

These are the six codons encoding Leucine

UUA, UUG, CUU, CUC, CUA, CUG

How many tRNAs minimal are needed for Leucine?

Three tRNAs are needed.

Aminoacyl-tRNA synthetase

Links amino acids to correct tRNA

What does the linking of amino acids to the correct tRNA result in

Result in activated (or charged) tRNA (e.g. methionyl tRNAMet)

• ATP driven

• high energy ester bond formed

What is the specific sequence that a eukaryotic ribosome scans for to find the correct AUG start codon?

The Kozak sequence (ACCAUGG)

How can some viral mRNAs initiate translation without binding to the 5' cap?

• By using an IRES (Internal Ribosome Entry Site).

Outline the 5 main steps of Eukaryotic Translation Initiation.

1. tRNA Binding: Methionyl tRNAMet is bound by eIFs (eukaryotic initiation factors).

2. Subunit Assembly: The tRNAMet-eIF complex binds to the small ribosomal subunit (40S).

3. mRNA Attachment: This complex binds to the 5' cap of the mRNA (or IRES for some viruses).

4. Scanning: The complex scans the mRNA until it finds the Kozak sequence (ACCAUGG).

5. Final Assembly: tRNA base-pairs with the AUG start codon, and the large subunit (60S) binds to complete the ribosome.

Eukaryotic initiation factors

are proteins that facilitate the initiation of translation by assisting in the assembly of the ribosome with the mRNA and tRNA.

What are the three sequential steps of the Translation Elongation cycle?

1. Aminoacyl tRNA binding: A new tRNA enters the A site.

2. Peptide bond formation: The growing chain is transferred to the new amino acid.

3. Translocation: The ribosome moves down the mRNA by one codon.

Elongation Factors (EFs)

Elongation factors (EFs) are essential proteins that assist in the translation elongation process, ensuring efficient and accurate incorporation of amino acids into the growing polypeptide chain.

When a stop codon enters the A site, what binds to it?

A Release Factor (RF).

• Termination is unique because a protein (Release Factor) recognizes the codon instead of an anticodon on a tRNA.

What provides the energy for the Release Factor to dissociate the translation complex?

GTP hydrolysis (GTP → GDP + Pi​).

What are the four main components released at the end of Translation Termination?

1. The completed Polypeptide (protein) chain.

2. The mRNA strand.

3. The Free tRNA.

4. The Ribosomal subunits(which dissociate to be reused).

Kanamycin is an antibiotic that binds to bacterial ribosomes and allows diverse tRNA with any anticodons to bind in the A site. What effect does kanamycin therefore have on bacterial translation?

a. It prevents the components of the translational apparatus from

coming together with a molecule of mRNA.

b. It causes incorporation of incorrect amino acids into a polypeptide.

c. It causes premature termination of translation.

d. It causes both effects b and c.

b. It causes incorporation of incorrect amino acids into a polypeptide.

A nonsense mutation is one that causes _____.

a. incorporation of the wrong amino acid at one

position in a polypeptide

b. premature termination of translation

c. either a or b.

b. premature termination of translation.

List two mechanisms the cell uses to counter Nonsense Mutations (premature stop codons).

1. Suppressor tRNA: A mutated tRNA that "reads through" a stop codon as an amino acid.

2. Nonsense-Mediated Decay (NMD): Destroys mRNA containing premature stops before they can be translated multiple times.

During Nonsense-Mediated Decay (NMD), what specific complex helps identify a stop codon as "premature" rather than normal?

The Exon Junction Complex (EJC).

• If a stop codon appears "upstream" of an EJC, the cell knows it's a mistake.

What is a Non-stop Mutation, and what is its effect on the Open Reading Frame (ORF)?

It is a mutation where a normal stop codon is changed to a regular (amino acid) codon.

• Translation "reads through" the boundary, adding extra, incorrect amino acids until the end of the transcript.

How do eukaryotes handle a ribosome that is stalled at the end of an mRNA that lacks a stop codon?

Nonstop Decay: An RNA-degrading enzyme binds to the empty A site of the stalled ribosome and degrades the defective mRNA.

• Contrast the "A site" state in Normal Termination vs. Nonstop Decay

• Normal: A Stop Codon is in the A site, and a Release Factor binds.

• Nonstop Decay: The A site is empty (it's at the very end of the mRNA), and an RNA degrading enzyme binds.

What are Exon Junction Complexes (EJCs), and where are they deposited on an mRNA strand?

They are protein complexes deposited precisely at the junctions where two exons were spliced together (after introns were removed).

During a normal round of translation, what happens to the Exon Junction Complexes (EJCs) as the ribosome moves along the mRNA?

The moving ribosome displaces (knocks off) all the EJCs as it moves toward the true stop codon.

How does the cell use EJCs to identify a premature stop codon (nonsense mutation)?

If a ribosome hits a stop codon while there are still EJCs attached further downstream, the cell recognizes the stop as "premature."

What is the fate of an mRNA where a ribosome stops "upstream" of an EJC?

It triggers Nonsense-Mediated Decay (NMD), which degrades the mRNA so it can't produce more truncated proteins.

Chaperonins

are a class of protein chaperones that assist in the proper folding of other proteins by providing a protective environment.

What does the misfolding of proteins lead to?

lead to abnormal protein aggregation and some lead to serious diseases

– Alzheimer’s

– Mad cow disease

Where does the initiation of translation occur for all proteins?

On free cytosolic ribosomes.

What is Cotranslational Import, and what are the typical final destinations for these proteins?

It is when a ribosome attaches to the ER membrane while still synthesizing the protein.

• ER lumen, Golgi complex, Lysosomes, Plasma membrane, or Secretory vesicles (secreted out of the cell).

What is Posttranslational Import, and what are the typical final destinations for these proteins?

The protein is fully completed by a free ribosome in the cytosol before being sorted.

• Destinations: Nucleus, Mitochondria, Chloroplasts, Peroxisomes, or remaining in the Cytosol.

If a protein is destined for the Plasma Membrane, is it likely made by a free ribosome or an ER-bound ribosome?

ER-bound ribosome (via the Cotranslational/Secretory pathway).

If a protein is destined for the Mitochondria, is it imported during or after translation?

After translation (Posttranslational Import).

What is the role of the Signal Recognition Particle (SRP) in cotranslational import?

It binds to the ER signal sequence on the emerging protein, blocks further translation, and escorts the ribosome to the ER membrane.

Sequence the following components in order of binding: SRP receptor, Translocon, SRP, Signal Sequence.

1. Signal Sequence (emerges from ribosome)

2. SRP (binds the sequence)

3. SRP Receptor (docks the complex to the ER)

4. Translocon (the pore opens and translation resumes)

What happens to the ER signal sequence once the protein begins entering the ER lumen?

• It is cleaved off by an enzyme called Signal Peptidase.

Define the function of a Stop-transfer sequence in membrane protein synthesis.

It is a hydrophobic amino acid sequence that halts translocation through the pore, causing the protein to remain anchored in the ER membrane.

How does an Internal start-transfer sequence differ from a terminal signal sequence?

It is not at the very end of the protein and is not cleaved by signal peptidase; it acts as both a docking signal and a permanent membrane anchor.

If a protein has a terminal ER signal sequence followed later by a stop-transfer sequence, where will the N-terminusand C-terminus end up?

N-terminus: Inside the ER Lumen (because the signal was cleaved inside).

C-terminus: In the Cytosol (because the stop-transfer sequence "locked" it in the membrane).