10.3:
tRNA Activation
To decode an mRNA into a protein sequence, each tRNA molecule carrying an amino acid, recognizes the three-nucleotide codon sequence on the mRNA, that corresponds to the amino acid.
There are 61 distinct codon sequences that encode 20 amino acids present in a cell. A single amino acid is coded for by several different codons, with one tRNA carrying one amino acid.
During the pairing of the tRNA anticodon with the mRNA codon, once the first two positions are paired, the third base can pair to either of the purines or either of the pyrimidines. This “wobble base”, allows 20 tRNAs to decode 61 mRNA codons.
An amino acid is covalently attached to the 3’ end of its partner tRNA by a group of enzymes called aminoacyl tRNA synthetases. There are 20 different aminoacyl tRNA synthetases corresponding to 20 amino acids.
The catalytic reaction proceeds in two steps.
The first step is amino acid activation, where within the enzyme pocket, the amino acid reacts with an ATP to form an aminoacyl AMP synthetase intermediate.
In the second step of esterification, the activated amino acid is joined to a hydroxyl group at the 3’ terminus of the tRNA, forming the final aminoacyl-tRNA molecule.
If the enzyme binds the wrong amino acid, it can correct the mistake through a proofreading mechanism.
The correct amino acid has high affinity for the active site of the enzyme. Larger amino acids are rejected from the active site.
If an amino acid is similar in size to the correct one, before getting coupled to a tRNA, the incorrect aminoacyl AMPs are forced into a second editing pocket within the enzyme.
Because the dimensions of this editing site precisely fit the correct amino acid, incorrect amino acyl AMPs are hydrolyzed rather than being joined to the tRNA.
10.3:
tRNA Activation
Aminoacyl-tRNA synthetases are present in both eukaryotes and bacteria. Though eukaryotes have 20 different aminoacyl-tRNA synthetases to couple to 20 amino acids, many bacteria do not have genes for all of these aminoacyl-tRNA synthetases. Despite this, they still use all 20 amino acids to synthesize their proteins. For instance, some bacteria do not have the gene encoding the enzyme that couples glutamine with its partner tRNA. In these organisms, one enzyme adds glutamic acid to all of the glutamic acid tRNA molecules, as well as all of the glutamine tRNA molecules. Then, a second enzyme chemically modifies the glutamic acid into glutamine on the latter tRNA molecules, thus forming the proper pair.
The equal importance of tRNA and aminoacyl-tRNA synthetase was established by a series of experiments in which one amino acid was chemically converted into a different amino acid after being attached to its paired tRNA. In an in vitro protein synthesis experiment, these "hybrid" aminoacyl-tRNA molecules inserted the incorrect amino acid at every point in the peptide chain where that tRNA was used. The results showed that both the tRNA and the enzyme are equally required for proper translation of the amino acid sequence encoded by the mRNA.
In cells, aminoacyl-tRNA synthetases use structural and chemical complementarity to identify the correct tRNA that must be coupled to the amino acid bound at its active site. Most tRNA synthetases contain three adjacent nucleotide-binding pockets, each of which is complementary in shape and charge to a nucleotide in the anticodon. While these pockets recognize the specific nucleotides in the anticodon loop of the tRNA, additional amino acids interact with the amino acid-accepting arm, thus allowing the correct tRNA to fit into the synthesis site of the enzyme.
Suggested Reading
- Alberts, Bruce. "Molecular Biology of the Cell." (2016), Pgs 336-339.
- “PDB101: Molecule of the Month: Aminoacyl-TRNA Synthetases.” RCSB, PDB-101, Apr. 2001, pdb101.rcsb.org/motm/16.