The assembly of a new polypeptide from its constituent amino acids is governed by a trip let genetic code. Within an mRNA, the central nucleotide sequence that is used to make the polypeptide is scanned from 5′ to 3′ on the ribosome in groups of three nucleotides, called codons (the corresponding groups of three nucleotides on the sense strand of DNA are called triplets).
Each codon specifies an amino acid and the decoding process uses a collection of different tRNA molecules, each of which binds one type of amino acid. An amino acid–tRNA complex is known as an aminoacyl tRNA and is formed when a dedicated aminoacyl tRNA synthetase covalently links the required amino acid to the terminal adenosine in the conserved CCA trinucleotide at the 3′ end of the tRNA.
Each tRNA has its own anticodon, a trinucleotide at the center of the anticodon arm (see Figure 1) that provides the necessary specificity to interpret the genetic code. For an amino acid to be added to a growing polypeptide, the relevant codon of the mRNA molecule must be recognized by base pairing with a complementary anticodon on the appropriate aminoacyl tRNA molecule. This happens on the ribosome. The small ribosomal subunit binds the mRNA, while the large subunit has two sites for binding amino acyl tRNAs: a P (peptidyl) site and an A (aminoacyl) site (Figure 2).
Fig1. Extensive intramolecular base pairing and nucleoside modification in transfer RNA. The tRNAGly shown here has the classical cloverleaf tRNA structure, with three stem-loops (the D arm, the anticodon arm, and the TψC arm) plus a stretch of base pairing between the 5′ and 3′ terminal sequences (called the acceptor arm because the 3′ end is where an amino acid is attached). Note that different tRNAs tend to have the same number of base pairs in the stems of the different arms of their cloverleaf structure and that the anticodon at the center of the middle loop identifies the tRNA according to the amino acid it will bear. Nine of the 74 nucleotides have been subjected to a nucleoside modification, including four 5-methylcytidines (m5C) and one each of 1-methyladenosine (m1A), 2′-O-methyluridine (Um), 5,6-dihydrouridine (D), and pseudouridine (ψ). In addition, a uracil was methylated at its carbon 5 to give thymine (T).
Fig2. The genetic code is deciphered on ribosomes by codon anticodon recognition. (A) The large subunit (60S in eukaryotes) has two sites for binding an aminoacyl tRNA (a transfer RNA with its attached amino acid): the P (peptidyl) site and the A (aminoacyl) site. The small ribosomal subunit (40S in eukaryotes) binds mRNA, which is scanned along its 5′ UTR in a 5′ → 3′ direction until the start codon is identified, an AUG located within a larger consensus sequence (see text). An initiator tRNAMet carrying a methionine residue binds to the P site with its anticodon in register with the AUG start codon. (B) The appropriate aminoacyl tRNA is bound to the A site with its anticodon base pairing with the next codon (GGG in this case, specifying glycine). (C) The rRNA in the large subunit catalyzes peptide bond formation, resulting in the methionine detaching from its tRNA and being bound instead to the glycine attached to the tRNA held at the A site. (D) The ribosome translocates along the mRNA so that the tRNA bearing the Met-Gly dipeptide is bound by the P site. The next aminoacyl tRNA (here, carrying Tyr) binds to the A site in preparation for new peptide bond formation. (E) Peptide bond formation. The N atom of the amino group of the amino acid bound to the tRNA in the A site makes a nucleophilic attack on the carboxyl C atom of the amino acid held by the tRNA bound to the P site.
The cap at the 5′ end of messenger RNA molecules is important in initiating translation. It is recognized by certain key proteins that bind the small ribosomal subunit and these initiation factors hold the mRNA in place. In cap-dependent translation initiation, the ribosome scans the 5′ UTR of the mRNA in the 5′ → 3′ direction to find a suitable initiation codon, an AUG that is found within the Kozak consensus sequence 5′GCCPuCCAUGG3′ (where Pu = purine). The most important determinants are the G at position +4 (immediately following the AUG codon) and the purine (preferably A) at −3 (three nucleotides upstream of the AUG codon).
When a suitable initation codon is identified, an initiating tRNAMet with its attached methionine binds to the P site on the large ribosomal subunit so that its anticodon base-pairs with the AUG initiator codon on the mRNA (see Figure 2). Once this hap pens, the transcriptional reading frame is established and codons are interpreted as successive groups of three nucleotides continuing in the 5′ → 3′ direction downstream of the initiating AUG codon. An aminoacyl tRNA for the second codon (a tRNAGly to recognize GGG in the example of Figure 2) binds to the neighboring A site in the large subunit.
Once the P and A sites are occupied by aminoacyl tRNAs, the 28S rRNA within the large ribosomal subunit acts as a peptidyltransferase. (An RNA like this that works as an enzyme is said to be a ribozyme.) The 28S rRNA catalyzes formation of a peptide bond by a condensation reaction between the amino group of the amino acid held by the tRNA in the A site and the carboxyl group of the methionine held by the tRNAMet. The net result is to detach the initiator methionine from its tRNA and attach it to the second amino acid, forming a dipeptide (see Figure 2). Now without any attached amino acid, the tRNAMet migrates away from the P site and its place is taken by the tRNA with the attached dipeptide that formerly occupied the A site. The liberated A site is now filled by an aminoacyl tRNA carrying an anticodon that is complementary to the third codon, and a new peptide bond is formed to make a tripeptide, and so on.
After a ribosome initiates translation of an mRNA and then moves along the mRNA, other ribosomes can engage with the same mRNA. The resulting polyribosome structures (polysomes) make multiple copies of a polypeptide from the one mRNA molecule. Polypeptide chain elongation occurs until a termination codon is met. In the case of mRNA transcribed from nuclear genes, termination codons come in three varieties: UAA (ochre); UAG (amber); and UGA (opal), but there are some differences in the case of mitochondrial mRNA as described in the next section.
In response to a termination codon, a protein release factor enters the A site instead of an aminoacyl tRNA to signal that the polypeptide should disengage from the ribosome.
The completed polypeptide will then undergo processing that can include cleavage and modification of the side chains. Its backbone will have a free amino group at one end (the N-terminal end) and a free carboxyl group at the other end (C-terminal end).
