Initiation of Protein Synthesis

The protein synthetic machinery must select the appropriate starting points for mRNA reading and peptide bond formation. AUG is usually used as the starting codon, and essentially all proteins begin with a methionine. AUG is also the codon for methionine that occurs in the interior of a protein as well, so there must be a mechanism to distinguish between the two types of methionine codons.

The steps of initiation occur on the isolated small subunit (30S) of the prokaryotic ribosome. Ribosomes contain two subunits, a 30S and 50S subunit, which associate to form a 70S particle. (The S values refer to the rate at which each component sediments in the ultracentrifuge; they don't always add up.) In general, the 30S subunit is mostly involved in the decoding and tRNA‐mRNA interaction process, while the 50S subunit is involved in actual peptide bond synthesis. Ribosomal subunits are dissociated prior to the initiation reaction.

Translation is initiated at the 5′ end of mRNA. Because RNA is synthesized in a 5′‐3′ direction, a bacterial mRNA can start translation while the 3′ sequences are still being transcribed. This is important in several forms of biological control.

A special initiator tRNA, tRNA met I (I stands for initiator) is used for beginning protein synthesis. In bacteria, this initiator tRNA carries the modified amino acid N‐formylmethionine (fmet). The formylation reaction transfers the formyl group from formyl‐tetrahydrofolate to methionyl‐tRNA metI +. This initiator tRNA is used to recognize initiation codons; it does not insert met in response to an internal AUG codon. As a further safeguard, the formylation reaction ensures that the initiator methionine can only be at the amino terminus of the synthesized protein.

The decoding step of protein synthesis involves base‐pairing between mRNA codon and tRNA anticodon sequences. A further base‐pairing event between noncoding regions of mRNA and rRNA is required to select the proper reading frame and initiation codon. Bacterial mRNAs contain a purine‐rich sequence (called a ‘Shine‐Dalgarno’ or RBS, which is an abbreviation of Ribosome‐Binding Sequence) in the 5′ nontranslated region of the mRNA. This sequence is complementary to the 3′ end of the small subunit rRNA, 16S rRNA. See Figure  1.

                        Figure 1

After base‐pairing is established, protein synthesis starts with the first AUG downstream of the RBS. This feature of initiation is used as a form of translational control. Messenger RNAs with the greatest degree of RBS complementarity to 16S rRNA are translated most efficiently, presumably because they initiate more efficiently.

Several protein factors are involved in the initiation process. These factors aren't usually part of the ribosome; instead, they help form an active initiation complex. Initiation factor 3 (IF3) helps keep the 30S subunit dissociated from the 50S subunit and available for protein synthesis. IF1 binds to the isolated 30S subunit and helps form the complex between the RBS and 16S rRNA. IF2 forms a complex with fmet‐tRNA met I and GTP, releasing IF3. After the complex contains mRNA and initiator fmet‐tRNA, the following things occur: GTP is hydrolyzed to GDP, the initiation factors are released from the ribosome, and the 50S subunit associates with the complex to form an elongating ribosome, as shown in Figure  2.

                          Figure 2