BIOCHEMISTRY - L. Stryer - 1984

VOLUME 3

Part IV INFORMATION

CHAPTER 27. PROTEIN SYNTHESIS

27.15. Formation of the 70S Initiation Complex Places Formylmethionyl-tRNA in the P Site

Protein Synthesis begins with the assembly of mRNA, the 30S ribosomal subunit, and formylmethionyl-tRNA to form the 30S initiation complex (Fig. 27.20). GTP and three protein factors, designated IF-1, IF-2, and IF-3, are required for this process. One of these initiation factors, IF-3, participates in binding mRNA to the 30S subunit. Furthermore, IF-3 prevents the premature association of 50S and 30S subunits into an unproductive 70S complex lacking mRNA, whereas IF-1 and IF-2 facilitate the binding of the initiator tRNA to the mRNA-30S subunit complex.

Class="center">Fig. 27.20. Initiation stage of protein synthesis: Formation of the 30S initiation complex followed by the 70S initiation complex

Next, the 50S subunit joins the 30S initiation complex to yield the 70S initiation complex, a step accompanied by the Hydrolysis of bound GTP. The 70S initiation complex (Fig. 27.20) is now primed for the elongation phase of protein synthesis. The fMet-tRNA_f molecule occupies the P (peptidyl) site of the ribosome. The second ribosomal site for tRNA binding, the A (aminoacyl) site, is still vacant. The existence of distinct P and A sites was demonstrated through studies with puromycin, an antibiotic discussed below (Section 27.21). Crucially, fMet-tRNA_f is positioned so that its anticodon pairs with the initiation codon AUG (or GUG) on the mRNA. Thus, the reading frame is established by the specific interaction of the ribosome and fMet-tRNA with the mRNA. Recall that this interaction involves a purine-rich sequence located 5' to the initiation codon, which pairs with the 3' end of 16S rRNA within the 30S subunit. Elucidating this intricate mechanism for the precise orientation of the initiator tRNA raised a fascinating question: how could poly(U) and other synthetic Polypeptides lacking start signals be translated in the experiments used to crack METABOLISM/28.html">The Genetic Code (Section 26.3)? The answer is that, fortuitously, those experiments involved nonspecific Translation because the Mg²⁺ concentration in the reaction mixture was higher than that found in vivo.

27.16. Elongation Factor Tu Delivers Aminoacyl-tRNA to the A Site of the Ribosome

The elongation cycle of protein synthesis consists of three steps: (1) binding of aminoacyl-tRNA (codon recognition); (2) peptide bond formation; and (3) translocation. The cycle begins with the delivery of aminoacyl-tRNA to the vacant A site of the ribosome. The choice of the correct tRNA species depends on the mRNA codon currently exposed in the A site. The complementary aminoacyl-tRNA is delivered to the A site by a protein known as elongation factor EF-Tu. Once the aminoacyl-tRNA is correctly positioned on the ribosome, the GTP bound to EF-Tu is hydrolyzed. GDP remains tightly bound to EF-Tu until it is displaced by another elongation factor, EF-Ts. The resulting Tu-Ts complex dissociates upon the binding of GTP to Tu, generating a fresh Tu-GTP complex ready for the next round of elongation. This protein association and dissociation (Fig. 27.21) is driven in a repetitive cyclical manner by the energy of GTP hydrolysis.

Fig. 27.21. Reaction cycle involving elongation factor Tu

It is important to note that EF-Tu does not interact with fMet-tRNA_f, which is why the initiator tRNA is excluded from the A site. Conversely, Met-tRNA binds to EF-Tu just like any other aminoacyl-tRNA, which explains why internal AUG codons are not recognized by the initiator tRNA.

27.17. Peptide Bond Formation Is Followed by Translocation

We now have a complex in which aminoacyl-tRNA occupies the A site, while fMet-tRNA occupies the P site. Everything is set for peptide bond formation (Fig. 27.22). This reaction is catalyzed by peptidyl transferase, an enzyme that is an integral component of the 50S subunit. The activated formylmethionyl residue of fMet-tRNA_f (located in the P site) is transferred to the amino group of the aminoacyl-tRNA (in the A site) to form peptidyl-tRNA.

Fig. 27.22. Formation of a peptide bond

Following peptide bond formation, the deacylated tRNA occupies the P site, and the peptidyl-tRNA occupies the A site. The next phase of the elongation cycle is translocation, which involves three simultaneous movements: the deacylated tRNA leaves the P site, the peptidyl-tRNA shifts from the A site to the P site, and the mRNA advances by three NUCLEOTIDES. As a result, the next codon is properly positioned for reading. Translocation requires a third elongation factor, EF-G (also known as translocase). During translocation, the GTP bound to EF-G is hydrolyzed, which triggers the release of EF-G from the ribosome, allowing it to act catalytically. Translocation provides another striking example of directed motion powered by nucleoside triphosphate hydrolysis. Upon completion of translocation, the A site is vacated and ready to bind the next aminoacyl-tRNA, initiating a new elongation cycle (Fig. 27.23).

Fig. 27.23. Elongation stage of protein synthesis: binding of aminoacyl-tRNA, peptide bond formation, and translocation