- The context of a codon in mRNA refers to the fact that neighboring sequences may change the efficiency with which a codon is recognized by its aminoacyl-tRNA or is used to terminate protein synthesis.
- IF-2 binds the initiator fMet-tRNAf and allows it to enter the partial P site on the 30S subunit.
The meaning of the AUG and GUG codons depends on their context. When the AUG codon is used for initiation, it is read as formyl-methionine; when used within the coding region, it represents methionine. The meaning of the GUG codon is even more dependent on its location. When present as the first codon, it is read via the initiation reaction as formyl-methionine. Yet when present within a gene, it is read by Val-tRNA, one of the regular members of the tRNA set, to provide valine as required by the genetic code.
How is the context of AUG and GUG codons interpreted? Figure 6.14 illustrates the decisive role of the ribosome, in conjunction with accessory factors.
In an initiation complex, the small subunit alone is bound to mRNA. The initiation codon lies within the part of the P site carried by the small subunit. The only aminoacyl-tRNA that can become part of the initiation complex is the initiator, which has the unique property of being able to enter directly into the partial P site to recognize its codon.
When the large subunit joins the complex, the partial tRNA-binding sites are converted into the intact P and A sites. The initiator fMet-tRNAf occupies the P site. and the A site is available for entry of the aminoacyl-tRNA complementary to the second codon of the gene. The first peptide bond forms between the initiator and the next aminoacyl-tRNA.
Initiation prevails when an AUG (or GUG) codon lies within a ribosome-binding site, because only the initiator tRNA can enter the partial P site generated when the 30S subunit binds de novo to the mRNA. Internal reading prevails subsequently, when the codons are encountered by a ribosome that is continuing to translate an mRNA, because only the regular aminoacyl-tRNAs can enter the (complete) A site.
Accessory factors are critical in controlling the usage of aminoacyl-tRNAs. All aminoacyl-tRNAs associate with the ribosome by binding to an accessory factor. The factor used in initiation is IF-2 (see 6.4 Initiation in bacteria needs 30S subunits and accessory factors), and the corresponding factor used at elongation is EF-Tu (see 6.10 Elongation factor Tu loads aminoacyl-tRNA into the A site).
The initiation factor IF-2 places the initiator tRNA into the P site. By forming a complex specifically with fMet-tRNAf, IF-2 ensures that only the initiator tRNA, and none of the regular aminoacyl-tRNAs, participates in the initiation reaction. Conversely, EF-Tu, which places aminoacyl-tRNAs in the A site cannot bind fMet-tRNAf , which is therefore excluded from use during elongation.
An additional check on accuracy is made by IF-3, which stabilizes binding of the initiator tRNA by recognizing correct base pairing with the second and third bases of the AUG initiation codon.
Figure 6.15 details the series of events by which IF-2 places the fMet-tRNAf initiator in the P site. IF-2, bound to GTP, associates with the P site of the 30S subunit. At this point, the 30S subunit carries all the initiation factors. fMet-tRNAf. then binds to the IF-2 on the 30S subunit. IF-2 then transfers the tRNA into the partial P site.