Initiation codon: The initiating AUG is recognized by a special initiator tRNA (tRNAi). Recognition is facilitated by IF-2-GTP in prokaryotes and eIF-2-GTP (plus additional eIF) in eukaryotes. The charged tRNAi is the only tRNA recognized by (e)IF-2 and the only tRNA to go directly to the P site on the small subunit. [Note: Base modifications distinguish tRNAi from the tRNA used for internal AUG codons.] In bacteria and mitochondria, tRNAi carries an N-formylated methionine (fMet), as shown in Figure 32.11. After Met is attached to tRNAi, the formyl group is added by the enzyme transformylase, which uses N10-formyl tetrahydrofolate (see p. 267) as the carbon donor. In eukaryotes, tRNAi carries a Met that is not formylated. In both prokaryotic and eukaryotic cells, this N-terminal Met is usually removed before translation is completed. The large ribosomal subunit then joins the complex, and a functional ribosome is formed with the charged tRNAi in the P site. The A site is empty. [Note: Specific (e)IF function as anti-association factors and prevent premature addition of the large subunit.] The GTP on (e)IF-2 gets hydrolyzed to GDP. In eukaryotes, the guanine nucleotide exchange factor eIF-2B facilitates the reactivation of eIF-2-GDP through replacement of GDP by GTP.
B. Elongation
Elongation of the polypeptide involves the addition of amino acids to the carboxyl end of the growing chain. Delivery of the aminoacyl-tRNA whose codon appears next on the mRNA template in the ribosomal A site (a process known as decoding) is facilitated in E. coli by elongation factors EF-Tu-GTP and EF-Ts and requires GTP hydrolysis. [Note: In eukaryotes, comparable elongation factors are EF-1α-GTP and EF-1βγ. Both EF-Ts and EF-1βγ function in guanine nucleotide exchange.] Peptide-bond formation between the α-carboxyl group of the amino acid in the P site and the αamino group of the amino acid in the A site is catalyzed by peptidyltransferase, an activity intrinsic to an rRNA of the large subunit (Fig. 32.12). [Note: Because this rRNA catalyzes the reaction, it is a ribozyme (see p. 54).] After the peptide bond has been formed, the peptide on the tRNA at the P site is transferred to the amino acid on the tRNA at the A site, a process known as transpeptidation. The ribosome then advances three nucleotides toward the 3′-end of the mRNA. This process is known as translocation and, in prokaryotes, requires the participation of EF-G-GTP (eukaryotes use EF-2-GTP) and GTP hydrolysis. Translocation causes movement of the uncharged tRNA from the P to the E site for release and movement of the peptidyl-tRNA from the A to the P site. The process is repeated until a termination codon is encountered. [Note: Because of the length of most mRNA, more than one ribosome at a time can translate a message. Such a complex of one mRNA and a number of ribosomes is called a polysome, or polyribosome.]
C. Termination