Core Components of the Protein-Synthesizing System
The key difference between translation and other template-directed biosyntheses is that there is no direct complementary base pairing between the nucleotide template and the synthesized polypeptide. Translating the four-letter nucleotide language into the twenty-letter amino acid language requires a biological code and a complex molecular apparatus. This includes:
- mRNA — the informational template containing a sequence of codons.
- Amino acids — the building blocks for protein construction.
- tRNA — adaptor molecules featuring an acceptor (-CCA) end for amino acid attachment and an anticodon for mRNA recognition.
- Aminoacyl-tRNA synthetases — specific enzymes that catalyze the joining of amino acids to their corresponding tRNAs.
- Ribosomes — ribonucleoprotein complexes where the physical assembly of the polypeptide chain takes place.
- Protein factors — non-ribosomal initiation (IF), elongation (EF), and termination (RF) factors.
- Energy and cofactors — ATP and GTP molecules, as well as magnesium ions required to stabilize the ribosomal structure.
Amino Acid Activation
Before an amino acid can enter the ribosome, it must be activated. This process is carried out by aminoacyl-tRNA synthetases (there are 20 types of these enzymes in the cell—one for each proteinogenic amino acid).
Interestingly, the enzyme possesses absolute specificity for its amino acid, but only relative specificity for its tRNA. Due to the degeneracy of the genetic code, a single amino acid may be carried by several different isoacceptor tRNAs (which differ in their anticodon sequences but bind the same amino acid).
During the reaction, the enzyme attaches the alpha-carboxyl group of the amino acid to the 3'-OH end of the tRNA, utilizing energy from ATP.
Example of aspartate activation: Asp + tRNA + ATP → Asp-tRNA + AMP + pyrophosphate.
The specificity of the resulting aminoacyl-tRNA is typically denoted with a superscript, for example: Met-tRNA^Met.
Translation Initiation
Protein synthesis begins with the assembly of an active ribosome, a process involving more than 10 initiation factors (IF). The steps of initiation are:
- A preinitiation complex forms, consisting of the small ribosomal subunit (40S in eukaryotes), the initiator Met-tRNA^Met, IF factors, and GTP energy.
- The complex binds to the 5'-cap of the mRNA and scans along the transcript until it encounters the start codon AUG.
- The anticodon of Met-tRNA^Met undergoes complementary base pairing with the start codon.
- The large ribosomal subunit (60S) joins, GTP is hydrolyzed, and the initiation factors (IF) are released.
The outcome of initiation is an assembled 80S ribosome with two functional active sites: the P-site (peptidyl) is occupied by the initiator tRNA, while the A-site (aminoacyl) is completely vacant and ready to accept the next molecule.
Elongation (Chain Elongation)
The elongation cycle repeats iteratively and consists of three sequential steps:
- Binding of aminoacyl-tRNA. A new aminoacyl-tRNA enters the vacant A-site. A strict requirement is that its anticodon must be complementary to the mRNA codon. This process is mediated by the elongation factor EF1 and requires GTP hydrolysis.
- Peptide bond formation. Methionine (or the growing peptide chain) is transferred from the P-site to the amino group of the incoming amino acid in the A-site. This reaction is catalyzed by peptidyl transferase. Notably, the active site of this enzyme is formed by the ribosomal RNA of the large subunit, meaning it exhibits ribozyme activity. This forms a dipeptidyl-tRNA.
- Translocation. The ribosome moves downstream along the mRNA by exactly one codon (in the 5' to 3' direction). This requires elongation factor EF2 and GTP hydrolysis. As a result, the peptidyl-tRNA moves into the P-site, the deacylated tRNA exits the ribosome, and the A-site becomes vacant again.
Translation Termination
Polypeptide assembly is completed when one of the stop codons enters the A-site: UAG, UGA, or UAA.
Instead of tRNAs, specialized protein termination factors—RF1 and RF3 (named for their structural homology to prokaryotic release factors)—bind to the stop codon.
Catalyzed by peptidyl transferase, the completed polypeptide chain is hydrolytically cleaved from the final tRNA. Following the release of the protein, the ribosomal subunits dissociate, which also requires the expenditure of a GTP molecule.