Genes VII

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.

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 6.11 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 yet 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.

So there are two differences between the initiating and elongating Met-tRNAs: the tRNA moieties themselves are different; and the amino acids differ in the state of the amino group.

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 6.12 illustrates the decisive role of the ribosome.

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 initiator fMet-tRNA_f lies in the now-intact 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.

So 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. The 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.

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 bound by IF-2.
• 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.
• A series of 3 G PC 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.

The ability of aminoacyl-tRNAs to enter the ribosome is controlled by accessory factors. Figure 6.14 shows how the fMet-tRNA_f initiator enters the P site. The 30S subunit carries all the initiation factors, including IF-2, which is probably associated with the P site. IF-2 specifically forms a complex with fMet-tRNA_f. This complex places the tRNA in the partial P site.

By forming a complex specifically with fMet-tRNA_f, IF-2 ensures that only the initiator tRNA, and none of the regular aminoacyl-tRNAs, participates in the initiation reaction.

IF-2 remains part of the 30S subunit at this stage; it has a further role to play. This factor has a ribosome-dependent GTPase activity: it sponsors the hydrolysis of GTP in the presence of ribosomes, releasing the energy stored in the high-energy bond.

The GTP is hydrolyzed when the 50S subunit joins to generate a complete ribosome. The GTP cleavage could be involved in changing the conformation of the ribosome, so that the joined subunits are converted into an active 70S ribosome.

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.