The second stage in the process of protein synthesis is initiation. At this stage, all the components necessary for protein synthesis assemble: (1) mRNA; (2) the small and large subunits of the ribosome; (3) a set of three proteins called initiation factors; (4) initiator tRNA with N-formylmethionine attached (fMet-tRNA^fMet); and (5) guanosine triphosphate (GTP). Initiation comprises three major steps. First, mRNA binds to the small subunit of the ribosome. Second, initiator tRNA binds to the mRNA through base pairing between the codon and the anticodon. Third, the large ribosome joins the initiation complex. Let’s look at each of these steps more closely.

297

INITIATION IN BACTERIA The functional ribosome of bacteria exists as two subunits, the small 30S subunit and the large 50S subunit (Figure 11.9a). An mRNA molecule can bind to the small ribosome subunit only when the subunits are separate. Initiation factor 3 (IF-3) binds to the small subunit of the ribosome and prevents the large subunit from binding during initiation (Figure 11.9b). Another factor, initiation factor 1 (IF-1), enhances the disassociation of the large and small ribosomal subunits.

The initiator tRNA, fMet-tRNA^fMet, then attaches to the initiation codon (Figure 11.9c). This attachment requires initiation factor 2 (IF-2), which forms a complex with GTP.

At this point, the initiation complex consists of (1) the small subunit of the ribosome; (2) the mRNA; (3) the initiator tRNA with its amino acid (fMet-tRNA^fMet); (4) one molecule of GTP; and (5) several initiation factors. These components are collectively known as the 30S initiation complex (see Figure 11.9c). In the final step of initiation, the initiation factors disassociate from the small subunit, allowing the large subunit of the ribosome to join the initiation complex (Figure 11.9d). When the large subunit has joined the initiation complex, the complex is called the 70S initiation complex.

INITIATION IN EUKARYOTES Similar events take place in the initiation of translation in eukaryotic cells, but there are some important differences. In bacterial cells, sequences in 16S rRNA of the small subunit of the ribosome bind to the Shine–Dalgarno sequence in mRNA. No analogous consensus sequence exists in eukaryotic mRNA. Instead, the cap at the 5′ end of eukaryotic mRNA plays a critical role in the initiation of translation. The small subunit of the eukaryotic ribosome, initiation factors, and the initiator tRNA with its amino acid (Met-tRNA^Met) form an initiation complex that recognizes the cap and binds there. The initiation complex then moves along (scans) the mRNA until it locates the first AUG codon. The identification of the start codon is facilitated by the presence of a consensus sequence (called the Kozak sequence) that surrounds the start codon:

Another difference is that eukaryotic initiation requires at least seven initiation factors. The poly(A) tail at the 3′ end of eukaryotic mRNA also plays a role in the initiation of translation. During initiation, proteins that attach to the poly(A) tail interact with proteins that bind to the 5′ cap, enhancing the binding of the small subunit of the ribosome to the 5′ end of the mRNA. This interaction indicates that the 3′ end of the mRNA bends over and associates with the 5′ cap during the initiation of translation, forming a circular structure known as the closed loop (Figure 11.10).

298