A special initiator tRNA starts the polypeptide chain

KEY TERMS:

• The initiation codon is a special codon (usually AUG) used to start synthesis of a protein.
• N-formyl-methionyl-tRNA (tRNA_f^Met) is the aminoacyl-tRNA that initiates bacterial protein synthesis. The amino group of the methionine is formylated.
• tRNA_f^Met is the special RNA that is to initiate protein synthesis in bacteria. It mostly uses AUG, but can also respond to GUG and CUG.
• tRNA_m^Met inserts methionine at internal AUG codons.

KEY CONCEPTS:

• Protein synthesis starts with a methionine amino acid usually coded by AUG.
• Different methionine tRNAs are involved in initiation and elongation.
• The initiator tRNA has unique structural features that distinguish it from all other tRNAs.
• The NH₂ group of the methionine bound to bacterial initiator tRNA is formylated. 

Synthesis of all proteins starts with the same amino acid: methionine. The signal for initiating a polypeptide chain is a special initiation codon that marks the start of the reading frame. Usually the initiation codon is the triplet AUG, but in bacteria, GUG or UUG are also used.

The AUG codon represents methionine, and two types of tRNA can carry this amino acid. One is used for initiation, the other for recognizing AUG codons during elongation.

In bacteria and in eukaryotic organelles, the initiator tRNA carries a methionine residue that has been formylated on its amino group, forming a molecule of N-formyl-methionyl-tRNA. The tRNA is known as tRNA_f^Met. The name of the aminoacyl-tRNA is usually abbreviated to fMet-tRNA_f (Marcker and Sanger, 1964).

The initiator tRNA gains its modified amino acid in a two stage reaction. First, it is charged with the amino acid to generate Met-tRNA_f; then the formylation reaction shown in Figure 6.12 blocks the free NH₂ group. Although the blocked amino acid group would prevent the initiator from participating in chain elongation, it does not interfere with the ability to initiate a protein.

This tRNA is used only for initiation. It recognizes the codons AUG or GUG (occasionally UUG). The codons are not recognized equally well: the extent of initiation declines about half when AUG is replaced by GUG, and declines by about half again when UUG is employed.

The species responsible for recognizing AUG codons in internal locations is tRNA_m^Met. This tRNA responds only to internal AUG codons. Its methionine cannot be formylated.

What features distinguish the fMet-tRNA_f initiator and the Met-tRNA_m elongator? Some characteristic features of the tRNA sequence are important, as summarized in Figure 6.13. Some of these features are needed to prevent the initiator from being used in elongation, others are necessary for it to function in initiation:

• Formylation is not strictly necessary, because nonformylated Met-tRNA_f can function as an initiator, but it improves the efficiency with which the Met-tRNA_f is used, because it is one of the features recognized by the factor IF-2 that binds the initiator tRNA (Sundari et al., 1976).
• The bases that face one another at the last position of the stem to which the amino acid is connected are paired in all tRNAs except tRNA_f^Met. Mutations that create a base pair in this position of tRNA_f^Met allow it to function in elongation. The absence of this pair is therefore important in preventing tRNA_f^Met from being used in elongation. It is also needed for the formylation reaction (Lee, Seong, and RajBhandary, 1991).
• A series of 3 G·C pairs in the stem that precedes the loop containing the anticodon is unique to tRNA_f^Met. These base pairs are required to allow the fMet-tRNA_f to be inserted directly into the P site.

In bacteria and mitochondria, the formyl residue on the initiator methionine is removed by a specific deformylase enzyme to generate a normal NH₂ terminus. If methionine is to be the N-terminal amino acid of the protein, this is the only necessary step. In about half the proteins, the methionine at the terminus is removed by an aminopeptidase, creating a new terminus from R₂ (originally the second amino acid incorporated into the chain). When both steps are necessary, they occur sequentially. The removal reaction(s) occur rather rapidly, probably when the nascent polypeptide chain has reached a length of 15 amino acids.