Lecture 23: Translation-Quality Control

nonstop, stalls

Overview of Quality Control (Bacteria)

• Termination: Typically, RF1 or RF2 binds a stop codon, releases the peptide, and RF3 removes the factor.

• Recycling: RRF (Ribosome Recycling Factor) and EF-G split the ribosomal subunits after termination.

• The Problem: What happens when this process fails? If there is no stop codon (___ mRNA) or the ribosome __, these standard termination factors cannot bind, requiring alternative rescue mechanisms

toxic, persists, tmRNA

• Scenario: If an mRNA is truncated (broken) or lacks a stop codon, the ribosome reaches the very end of the RNA.

• The Consequence:

  1. Stalled Ribosome: The ribosome is stuck at the 3' end with a peptidyl-tRNA in the P-site and nothing in the A-site. It cannot terminate or recycle.

  2. Toxic Protein: The incomplete protein fragment might be misfolded and _____.

  3. Defective mRNA: The faulty mRNA ___ and could trap more ribosomes.

• The Solution: Bacteria use trans-translation (___) to solve all three problems simultaneously ³

ssrA, acceptor stem, T-arm, alanine, ORF, peptide, enter, signal

tmRNA Structure (SsrA)

• Visuals: Comparison of a standard tRNA (left) and tmRNA (right).

• Explanation:

  • tmRNA (Transfer-Messenger RNA): A specialized RNA molecule (encoded by the __ gene) that mimics both a tRNA and an mRNA.

  • tRNA-like Domain (TLD): The top part folds exactly like a tRNA (__ ___ and _-_) and is charged with ______. However, it lacks an anticodon arm.

  • mRNA-like Domain (MLD): The bottom part contains a short ___ encoding a ___ tag (ANDENYALAA).

  • Function: It acts as a tRNA to ____ the stalled ribosome and then as an mRNA to provide a termination ____.

Recognition, Transpeptidation, Template Switching, Tagging, Terminaiton

Trans-Translation Mechanism Overview

1. _____: SmpB (Small protein B) binds tmRNA and helps it recognize the stalled ribosome.

2. ________: The alanine from tmRNA is added to the stalled polypeptide chain.

3. ___ ___: The ribosome switches from the defective mRNA to the internal ORF of the tmRNA.

4. _____: The ribosome translates the tmRNA ORF, adding a degradation tag (ANDENYALAA) to the protein.

5. _____: The tmRNA ends with a stop codon, allowing normal termination and ribosome recycling

SmpB, stalled

Trans-Translation Mechanism Overview

1. Recognition: _____ (Small protein B) binds tmRNA and helps it recognize the ___ ribosome.

2. Transpeptidation: The alanine from tmRNA is added to the stalled polypeptide chain.

3. Template Switching: The ribosome switches from the defective mRNA to the internal ORF of the tmRNA.

4. Tagging: The ribosome translates the tmRNA ORF, adding a degradation tag (ANDENYALAA) to the protein.

5. Termination: The tmRNA ends with a stop codon, allowing normal termination and ribosome recycling

alanine, polypeptide

Trans-Translation Mechanism Overview

1. Recognition: SmpB (Small protein B) binds tmRNA and helps it recognize the stalled ribosome.

2. Transpeptidation: The __ from tmRNA is added to the stalled __ chain.

3. Template Switching: The ribosome switches from the defective mRNA to the internal ORF of the tmRNA.

4. Tagging: The ribosome translates the tmRNA ORF, adding a degradation tag (ANDENYALAA) to the protein.

5. Termination: The tmRNA ends with a stop codon, allowing normal termination and ribosome recycling

ORF

Trans-Translation Mechanism Overview

1. Recognition: SmpB (Small protein B) binds tmRNA and helps it recognize the stalled ribosome.

2. Transpeptidation: The alanine from tmRNA is added to the stalled polypeptide chain.

3. Template Switching: The ribosome switches from the defective mRNA to the internal ___ of the tmRNA.

4. Tagging: The ribosome translates the tmRNA ORF, adding a degradation tag (ANDENYALAA) to the protein.

5. Termination: The tmRNA ends with a stop codon, allowing normal termination and ribosome recycling

degradation

Trans-Translation Mechanism Overview

1. Recognition: SmpB (Small protein B) binds tmRNA and helps it recognize the stalled ribosome.

2. Transpeptidation: The alanine from tmRNA is added to the stalled polypeptide chain.

3. Template Switching: The ribosome switches from the defective mRNA to the internal ORF of the tmRNA.

4. Tagging: The ribosome translates the tmRNA ORF, adding a _____ tag (ANDENYALAA) to the protein.

5. Termination: The tmRNA ends with a stop codon, allowing normal termination and ribosome recycling

stop

Trans-Translation Mechanism Overview

1. Recognition: SmpB (Small protein B) binds tmRNA and helps it recognize the stalled ribosome.

2. Transpeptidation: The alanine from tmRNA is added to the stalled polypeptide chain.

3. Template Switching: The ribosome switches from the defective mRNA to the internal ORF of the tmRNA.

4. Tagging: The ribosome translates the tmRNA ORF, adding a degradation tag (ANDENYALAA) to the protein.

5. Termination: The tmRNA ends with a ___ codon, allowing normal termination and ribosome recycling

Entry, Transfer, Release, Tagging

trans-translation Mechanism Detail

• _____: The tmRNA-SmpB-EF-Tu-GTP complex enters the empty A-site of a stalled ribosome.

• ______: EF-Tu hydrolyzes GTP, and the nascent chain is transferred to the Alanine on tmRNA.

• _____: The defective mRNA is released and degraded by RNase R.

• _____: The tagged protein is recognized and destroyed by proteases like ClpXP or Lon. This cleans up the cellular "trash"

EF-Tu-GTP, A

trans-translation Mechanism Detail

• Entry: The tmRNA-SmpB-__-___-__ complex enters the empty _-site of a stalled ribosome.

• Transfer: EF-Tu hydrolyzes GTP, and the nascent chain is transferred to the Alanine on tmRNA.

• Release: The defective mRNA is released and degraded by RNase R.

• Tagging: The tagged protein is recognized and destroyed by proteases like ClpXP or Lon. This cleans up the cellular "trash"

GTP, tmRNA

trans-translation Mechanism Detail

• Entry: The tmRNA-SmpB-EF-Tu-GTP complex enters the empty A-site of a stalled ribosome.

• Transfer: EF-Tu hydrolyzes ___, and the nascent chain is transferred to the Alanine on ____.

• Release: The defective mRNA is released and degraded by RNase R.

• Tagging: The tagged protein is recognized and destroyed by proteases like ClpXP or Lon. This cleans up the cellular "trash"

RNase R

trans-translation Mechanism Detail

• Entry: The tmRNA-SmpB-EF-Tu-GTP complex enters the empty A-site of a stalled ribosome.

• Transfer: EF-Tu hydrolyzes GTP, and the nascent chain is transferred to the Alanine on tmRNA.

• Release: The defective mRNA is released and degraded by ____ _.

• Tagging: The tagged protein is recognized and destroyed by proteases like ClpXP or Lon. This cleans up the cellular "trash"

ClpXP, Lon

trans-translation Mechanism Detail

• Entry: The tmRNA-SmpB-EF-Tu-GTP complex enters the empty A-site of a stalled ribosome.

• Transfer: EF-Tu hydrolyzes GTP, and the nascent chain is transferred to the Alanine on tmRNA.

• Release: The defective mRNA is released and degraded by RNase R.

• Tagging: The tagged protein is recognized and destroyed by proteases like ___ or ___. This cleans up the cellular "trash"

barrel, tmRNA, rescue

SmpB Structure and Tail

• SmpB Core: A beta-____ structure that binds the tRNA-like domain of ___.

• C-terminal Tail: An unstructured, flexible tail that is essential for function.

• Mutants: Deleting or mutating this tail (e.g., stopping at 132 or 148) destroys SmpB's ability to support ribosome ___, proving the tail is critical for function

degradation

tmRNA Secondary Structure: Tag Sequence: The sequence A-N-D-E-N-Y-A-L-A-A is explicitly shown in the coding region. This is the "__ signal" added to stalled proteins

A, peptidyl

Tail Mutants Defect

• Visuals:

  • (Left): Western blot showing tagged proteins. Wild-type (WT) SmpB supports tagging (dark smear), while tail-truncated mutants (SmpB132-155) do not.

  • (Right): Mechanism diagram showing the blockage.

• Explanation:

  • The Defect: Without the C-terminal tail, the tmRNA-SmpB complex can bind the ribosome but cannot accommodate into the _-site or catalyze __ transfer.

  • Role: The tail likely acts as a "feeler" or stabilizer that probes the mRNA channel, ensuring the A-site is truly empty (stalled) before allowing rescue to proceed

accomodation, stem-loop

Tail Function Summary

• Visuals: Text and diagram relating the tail to function.

• Explanation:

  • The C-terminal tail is required for ____ (moving the tRNA into position for chemistry).

  • It likely mimics the anticodon __-__ of a normal tRNA, filling the space in the decoding center that would usually be occupied by a codon-anticodon pair. This is another example of molecular mimicry

Glycine 132, Lysines, Arginines

Structural Basis of SmpB Binding

• Visuals: Ribbon diagram of SmpB showing residues K131, G132, K133.

• Explanation:

  • Hinge: ___ __ acts as a hinge, allowing the C-terminal tail to swing and move. This flexibility is crucial for the dynamic process of ribosome binding and accommodation.

  • Basic Residues: ___ (K) and __ (R) in the tail are positively charged, likely interacting with the negatively charged rRNA in the decoding center

helix

Cryo-EM of SmpB on Ribosome

• The C-terminal tail forms a __ inside the ribosome's mRNA channel, effectively substituting for the mRNA that should be there.

• Some structures suggest a second SmpB molecule might bind to facilitate the process, though the single SmpB model is the canonical view for the initial step

before, probes

Assembly Models

• Visuals: Models proposing how the complex assembles (Single vs. Two SmpBs).

• Explanation:

  • The scientific community has debated whether one or two SmpB proteins are involved.

  • Current Consensus: SmpB binds tmRNA ___ entering the ribosome. The tail of SmpB ___ the decoding center to verify the ribosome is stalled (empty A-site) .

stalled

Affinity Capture Experiment

• Explanation:

  • Setup: A "Nonstop" mRNA (lacking a stop codon) is used to stall ribosomes.

  • Tag: The mRNA has a His6-tag or biotin tag.

  • Capture: Ribosomes stalled on this mRNA are pulled down using affinity beads.

  • Result: Western blot shows SmpB is highly enriched on these ___ ribosomes compared to total ribosomes, proving SmpB specifically targets stalled complexes

stalled, intermediates

Sucrose Gradient Analysis

• Visuals: Sucrose gradient profile separating 30S, 50S, 70S, and polysomes.

• Explanation:

  • This technique separates ribosomal particles by size (density).

  • 70S Peak: Represents monosomes (single ribosomes). This is where the ___ complexes and the tmRNA rescue ___ are found

tightly, weakly, active

Explanation:

• Salt Sensitivity: High salt (300mM) disrupts weak interactions.

• Result: SmpB binds tmRNA very ___ (stable at high salt) but binds the ribosome __ on its own. This ensures SmpB doesn't clog up __ ribosomes; it only enters as a complex with tmRNA

peptidyl-tRNA, mRNA, empty

Empty A-site: The key signal for rescue is a ribosome with a __-___ in the P-site but no ____ in the A-site (because the mRNA ends).

Sensor: The SmpB tail senses this __ channel. If mRNA were present, it would block SmpB binding (steric hindrance) .

His22, His136

• Standard Decoding: A1492/A1493 flip out to read the codon-anticodon helix.

• Rescue: SmpB residues (____, ___) interact with these same nucleotides. SmpB tricks the ribosome into thinking a valid tRNA has bound, triggering the "closed" conformation required for transpeptidation

trans-translation, ArfA/ArfB, spontaneous

A stalled ribosome can be resolved in multiple ways:

1. tmRNA (__-______): The main pathway for nonstop mRNA. Releases ribosome, tags protein for degradation (ClpXP/Lon), degrades mRNA (RNase R).

2. ____/_____: Alternative rescue factors (backup systems) if tmRNA is overwhelmed or absent.

3. _____: Rare hydrolysis (very slow) .

E, RF2, A, A, peptide, GGQ

lternative Rescue Factors (ArfA/ArfB)

• ArfA: Binds the _-site and recruits __ to the _-site to terminate the stalled ribosome, even without a stop codon.

• ArfB: A "molecular crowbar" that binds the _-site and directly catalyzes hydrolysis of the ____, independent of release factors. It mimics the __ motif of release factors .

decoding, false, RF2

ArfA and RF2

Mechanism: ArfA binds in the ____ center and creates a "__" stop codon surface. It forces ___ to bind and release the peptide, rescuing the ribosome.

ArfA, ArfB

Dependency: __ requires RF2; __ acts alone

separated, Nonsense-mediated decay, nonstop decay, no-go decay

Eukaryotes have more complex quality control because transcription and translation are ______.

NMD (___-______ ______): Detects premature stop codons.

NSD (______ ________): Detects ribosomes stalled at the 3' end (no stop codon).

NGD (___-_____ ______): Detects ribosomes stalled in the middle of mRNA (hairpins, damage)

premature, 3’ end, middle

Eukaryotes have more complex quality control because transcription and translation are separated.

• NMD (Nonsense-Mediated Decay): Detects ____ stop codons.

• NSD (Nonstop Decay): Detects ribosomes stalled at the ____ _____ (no stop codon).

• NGD (No-Go Decay): Detects ribosomes stalled in the __ of mRNA (hairpins, damage)

tmRNA, RQC, Rqc2, alanine, threonine, CAT, ubiquitination

• Comparison of bacterial vs. eukaryotic rescue.

• Explanation:

  • Bacteria: Use ___ to add a degradation tag (ANDENYALAA) to the peptide.

  • Eukaryotes: Use the ____ (Ribosome Quality Control) complex. Instead of a nucleic acid template, a protein (____) recruits ___ and ____ tRNAs to add a "__ tail" (C-terminal Ala/Thr) to the stalled protein. This CAT tail signals for ____ and degradation

Dom34, Hbs1, A

Eukaryotic Termination/Recycling

• Visuals: Standard termination (eRF1/eRF3) vs. rescue.

• Explanation:

  • Standard: eRF1 recognizes the stop codon, eRF3 helps. ABCE1 splits the ribosome.

  • Rescue: If the ribosome stalls (e.g., no stop codon), the factors ___ (mimics eRF1) and ___ (mimics eRF3) bind the empty _-site to trigger dissociation

polyA tail, subunits, degraded, Ski7

Nonstop Decay Pathway

• Visuals: Pathway showing Dom34/Hbs1 action.

• Explanation:

  1. Ribosome stalls at the _____ ____

  2. Dom34/Hbs1 bind and split the ribosome ____.

  3. Exosome: The mRNA is ___ 3'-5'.

  4. ____: An alternative factor in yeast that also recruits the exosome .

60S, Ltn1, Rqc2, Cdc48

RQC Complex Action

• Visuals: Diagram of Ltn1 and Rqc2 acting on the 60S subunit.

• Explanation:

  • Once the ribosome is split, the ____ subunit still holds the stuck peptidyl-tRNA.

  • ___ (E3 Ligase): Binds the 60S and ubiquitinates the nascent chain.

  • __ (NEMF): Stabilizes Ltn1 and adds the "CAT tail" (Alanine/Threonine extension) to push the protein out of the tunnel so Ltn1 can reach exposed lysines to tag them.

  • ___: Extracts the ubiquitinated protein for proteasomal degradation

peptidyl-tRNA, ubiquitinates, CAT, tunnel, Ltn1, proteasomal

RQC Complex Action

• Visuals: Diagram of Ltn1 and Rqc2 acting on the 60S subunit.

• Explanation:

  • Once the ribosome is split, the 60S subunit still holds the stuck ___-____.

  • Ltn1 (E3 Ligase): Binds the 60S and ___ the nascent chain.

  • Rqc2 (NEMF): Stabilizes Ltn1 and adds the "____ tail" (Alanine/Threonine extension) to push the protein out of the _____ so _____ can reach exposed lysines to tag them.

  • Cdc48: Extracts the ubiquitinated protein for _____ degradation

ribosome, protein

Evolutionary Comparison

• Visuals: Table comparing Bacteria, Archaea, and Eukarya.

• Explanation:

  • Bacteria: tmRNA (SsrA) + SmpB.

  • Eukaryotes: Dom34/Hbs1 (splitting) + RQC (tagging/degradation).

  • Convergent Evolution: Different proteins, same goal: rescue the ____ and destroy the toxic half-____.

termination, exon junction complex

Nonsense Mediated Decay (NMD)

• Target: mRNAs with a Premature _____ Codon (PTC).

• Purpose: Prevents production of truncated, dominant-negative proteins.

• Mechanism: Relies on the ribosome sensing a stop codon upstream of an ____ _______ __ (EJC)

UPF, eRF1/3

NMD Models

• Visuals: Diagram of EJC-dependent vs 3' UTR-dependent NMD.

• Explanation:

  • Normal Termination: The stop codon is close to the poly(A) tail; PABP interacts with eRF3 to terminate efficiently.

  • Premature Termination: The stop codon is far upstream. The ribosome stops, but PABP is too far away. Instead, the ribosome encounters a downstream Exon Junction Complex (EJC).

  • Trigger: Interaction between _____ proteins (recruited by EJC) and the termination factors (____) triggers decapping and rapid mRNA degradation

displaces, bound

Ribosome Displacement

• Explanation:

  • In normal translation, the ribosome moves along the mRNA and ____ all EJCs.

  • If a stop codon occurs before an EJC, the EJC remains ____. This "persistent EJC" recruits UPF1/2/3 and signals "Error! Premature Stop!" leading to mRNA destruction

recognition, decapping, exonuclease, permanently

NMD Degradation Steps

1. ______: UPF1 activates.

2. _______: The 5' cap is removed.

3. ______: Xrn1 degrades the RNA 5' → 3’.

• This ensures the faulty mRNA is _____ removed from the pool

UPF1, 5’ cap, Xrn

NMD Degradation Steps

1. Recognition: ____ activates.

2. Decapping: The __ __ is removed.

3. Exonuclease: ___ degrades the RNA 5' → 3’.

• This ensures the faulty mRNA is permanently removed from the pool

nonsense, inhibiting

Clinical Relevance

• Many genetic diseases (Cystic Fibrosis, Duchenne Muscular Dystrophy) are caused by ____ mutations (PTCs).

• Therapy: Drugs like Gentamicin or Ataluren try to force the ribosome to "read through" the stop codon to make full-length protein.

• Problem: NMD fights this by destroying the mRNA before it can be read through. ______ NMD could enhance these therapies

combining

Dual Therapy: _____ a "read-through" drug (like an aminoglycoside) with an "NMD inhibitor" could treat genetic diseases more effectively by saving the mRNA and allowing protein production