9.4
Improving Translational Accuracy
A single eukaryotic mRNA is translated to yield a typical-sized protein in about 30–60 seconds.
During this process, translation accuracy is maintained by elongation factors, EF-Tu and EF-G in bacteria, and EF1 and EF2 in eukaryotes.
During translation, EF-Tu associates with GTP and aminoacyl-tRNA. Together, they bind the A site of the ribosome to form a codon-anticodon duplex.
Each codon–anticodon interaction is checked twice.
The first checkpoint is the complementary base pairing between the mRNA codon and the tRNA anticodon.
A correct codon–anticodon match binds more tightly than an incorrect tRNA, which will dissociate.
At the second checkpoint, part of the 16S rRNA in the small ribosomal subunit folds around the paired bases forming a network of ribosomal contacts that are specific for each position of the codon–anticodon.
A mismatched tRNA fails to make ribosomal contacts, and dissociates, while the correct tRNA changes the conformation of the catalytic center within the ribosome. This is called an induced fit.
This causes EF-Tu to hydrolyze GTP and dissociate from the ribosome. The released aminoacyl-tRNA can now participate in translation.
If an aminoacyl-tRNA escapes proofreading and is added to the growing polypeptide chain, a codon‒anticodon mismatch in the P site of the ribosome causes an increased rate of error in the A site.
With successive rounds of amino acid misincorporation, the faulty polypeptide chain is prematurely terminated by release factors and the flawed protein is degraded.
Base complementarity between the three base pairs of mRNA codon and the tRNA anticodon is not a failsafe mechanism. Inaccuracies can range from a single mismatch to no correct base pairing at all. The free energy difference between the correct and nearly correct base pairs can be as small as 3 kcal/ mol. With complementarity being the only proofreading step, the estimated error frequency would be one wrong amino acid in every 100 amino acids incorporated. However, error frequencies observed in organisms are significantly lower.
The high level of accuracy is guaranteed by two additional proofreading steps involving two principles from enzyme-substrate interactions.
Within the ribosome, the peptidyl transferase center (PTC) catalyzes the covalent bond formation between amino acids to form a polypeptide chain. Like any other enzyme, the PTC also has an active site that discriminates between substrates based on their molecular structure. Residues from the 16S rRNA of the small ribosomal subunit form hydrogen bonds with the base and backbone atoms of the codon-anticodon duplex. Only the correct tRNA can induce conformational changes in the PTC, which carries out the catalysis.
The second step, called kinetic proofreading, occurs after the irreversible dissociation of EF-Tu·GDP from the ribosome. GTP hydrolysis marks the start of a short time delay during which the aminoacyl-tRNA moves into the active site of PTC for catalysis. During this delay, any incorrect codon-anticodon pairs that slipped through the induced-fit step are more likely to dissociate than correct pairs. The reason for this is that the wrong tRNA makes weaker base pairs with the codon, and the time delay is longer for incorrect than correct matches.
A single eukaryotic mRNA is translated to yield a typical-sized protein in about 30–60 seconds.
During this process, translation accuracy is maintained by elongation factors, EF-Tu and EF-G in bacteria, and EF1 and EF2 in eukaryotes.
During translation, EF-Tu associates with GTP and aminoacyl-tRNA. Together, they bind the A site of the ribosome to form a codon-anticodon duplex.
Each codon–anticodon interaction is checked twice.
The first checkpoint is the complementary base pairing between the mRNA codon and the tRNA anticodon.
A correct codon–anticodon match binds more tightly than an incorrect tRNA, which will dissociate.
At the second checkpoint, part of the 16S rRNA in the small ribosomal subunit folds around the paired bases forming a network of ribosomal contacts that are specific for each position of the codon–anticodon.
A mismatched tRNA fails to make ribosomal contacts, and dissociates, while the correct tRNA changes the conformation of the catalytic center within the ribosome. This is called an induced fit.
This causes EF-Tu to hydrolyze GTP and dissociate from the ribosome. The released aminoacyl-tRNA can now participate in translation.
If an aminoacyl-tRNA escapes proofreading and is added to the growing polypeptide chain, a codon‒anticodon mismatch in the P site of the ribosome causes an increased rate of error in the A site.
With successive rounds of amino acid misincorporation, the faulty polypeptide chain is prematurely terminated by release factors and the flawed protein is degraded.
- Codon - A set of three nucleotides in DNA or RNA that codes for a specific amino acid.
- Anticodon - A set of three nucleotides in a tRNA molecule that pairs with a specific codon in mRNA.
- Kinetic Proofreading - A mechanism that increases the accuracy of processes such as DNA replication and protein synthesis.
- Peptidyl Transferase - An enzymatic site of the ribosome that carries out protein synthesis.
- Translation Proofreading - The process that ensures the accuracy of protein synthesis by checking that the correct tRNA has paired with the mRNA codon.
- Define Codon vs Anticodon - Understand the pairing between mRNA and tRNA (e.g., codon, anticodon).
- Contrast Codon and Anticodon - Learn their roles and differences (e.g., mRNA synthesis, tRNA pairing).
- Explore Kinetic Proofreading Examples - Look at how this process works (e.g., during protein synthesis).
- Explain the Peptidyl Transferase Mechanism - Understand its role in protein synthesis.
- Apply Translation Proofreading in Context - Learn how it ensures the accuracy of protein synthesis.
- What is the difference between a codon and an anticodon?
- What is kinetic proofreading and how does it work in protein synthesis?
- How does the peptidyl transferase mechanism contribute to protein synthesis?
From Chapter 9:
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9.4 : Improving Translational Accuracy
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9.2 : tRNA Activation
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