During the first stage of translation, the small and large ribosomal subunits assemble around an mRNA that has a Met-tRNA_i^Met correctly positioned at the start codon in the ribosomal P site. In eukaryotes, the assembly of this complex is mediated by a special set of proteins known as eukaryotic translation initiation factors (eIFs). As each individual component joins the complex, it is guided by interactions with specific eIFs. Several of the initiation factors bind GTP, and the hydrolysis of GTP to GDP functions as a proofreading switch that allows subsequent steps to proceed only if the preceding step has occurred correctly. Before GTP hydrolysis, the complex is unstable, allowing dissociation of the components and a second attempt at complex formation until the correct complex assembles, resulting in GTP hydrolysis and stabilization of the appropriate complex.

Considerable progress has been made in the past few years in understanding translation initiation in vertebrates. The current model for initiation of translation in vertebrates is depicted in Figure 5-24. Large and small ribosomal subunits released from a previous round of translation are kept apart by the binding of eIFs 1, 1A, and 3 to the small 40S subunit (Figure 5-24, top). The first step of translation initiation is formation of a 43S preinitiation complex. This preinitiation complex is formed when the 40S subunit with eIFs 1, 1A, and 3 associates with eIF5 and a ternary (three-part) complex consisting of the Met-tRNA_i^Met and eIF2 bound to GTP (Figure 5-24, steps 1 and 2). The initiation factor eIF2 alternates between association with GTP and GDP; it can bind Met-tRNA_i^Met only when it is associated with GTP. Cells can inhibit protein synthesis by phosphorylating a serine residue on the eIF2 bound to GDP; the phosphorylated complex is unable to exchange the bound GDP for GTP and cannot bind Met-tRNA_i^Met so protein synthesis cannot occur.

The mRNA to be translated is bound by the multisubunit eIF4 complex, which interacts with both the 5′ cap and the cytoplasmic poly(A)-binding protein (PABPC) bound in multiple copies to the mRNA poly(A) tail. Both interactions are required for translation of most mRNAs. This binding results in the formation of a circular complex (Figure 5-24, step 3). The eIF4 cap-binding complex consists of several subunits with different functions. The eIF4E subunit binds the 5′ cap on mRNAs (see Figure 5-14). The large eIF4G subunit binds cooperatively to several PABPC proteins bound to the mRNA poly(A) tail, and also forms a scaffold to which the other eIF4 subunits bind. The mRNA-eIF4 complex then associates with the preinitiation complex through an interaction between eIF4G and eIF3 (step 4).

The initiation complex then slides along, or scans, the associated mRNA as the helicase activity of eIF4A, stimulated by eIF4B, uses energy from ATP hydrolysis to unwind the RNA secondary structure (step 5). Scanning stops when the tRNA_i^Met anticodon recognizes the start codon, which is the first AUG downstream from the 5′ end in most eukaryotic mRNAs. Recognition of the start codon leads to hydrolysis of the GTP associated with eIF2, an irreversible step that prevents further scanning, resulting in formation of the 48S initiation complex (step 6). This commitment to the correct initiation codon is facilitated by eIF5, an eIF2 GTPase–activating protein (GAP, see Figure 3-34). Selection of the initiating AUG is facilitated by specific surrounding nucleotides called the Kozak sequence, for Marilyn Kozak, who defined it: (5′)ACCAUGG(3′). The A preceding the AUG (in bold) and the G immediately following it are the most important nucleotides affecting translation initiation efficiency.

Association of the large (60S) subunit with the small subunit, which is mediated by eIF5B bound to GTP, results in displacement of many of the initiation factors (step 7). Correct association between the ribosomal subunits results in hydrolysis of the eIF5B-bound GTP to GDP and the release of eIF5B-GDP and eIF1A (step 8), completing the formation of an 80S initiation complex. Coupling of the ribosome subunit–joining reaction to GTP hydrolysis by eIF5B allows the initiation process to continue only when the subunit interaction has occurred correctly. It also makes this an irreversible step, so that the ribosomal subunits do not dissociate until the entire mRNA is translated and protein synthesis is terminated.

The eukaryotic protein-synthesizing machinery begins translation of most cellular mRNAs within about 100 nucleotides of the 5′-capped end as just described. However, some cellular mRNAs contain an internal ribosome entry site (IRES) located far downstream from the 5′ end. It is thought that cellular IRESs form RNA structures that interact with a complex of eIF4A and eIF4G, which then associates with eIF3 bound to a 40S subunit with eIF1 and eIF1A. This assembly then binds an eIF2 ternary complex to assemble an initiation complex directly on a neighboring AUG codon. In addition, translation of some positive-stranded viral RNAs, which lack a 5′ cap, is initiated at viral IRES sequences. These RNAs fall into different classes depending on how many of the standard eIFs are required for initiation. In the case of cricket paralysis virus, the 200-nt-long IRES folds into a complex structure that interacts directly with the 40S ribosomal subunit and leads to initiation without any of the eIFs or even the initiator Met-tRNA_i^Met !

192

In bacteria, binding of the small ribosomal subunit to an initiation site occurs by a different mechanism that allows initiation at internal sites in the polycistronic mRNAs transcribed from operons. In bacterial mRNAs, a ∼6-bp sequence complementary to the 3′ end of the small rRNA precedes the AUG start codon by 4–7 nucleotides. Base pairing between this sequence in the mRNA, called the Shine-Dalgarno sequence after its discoverers, and the small rRNA places the small ribosomal subunit in the proper position for initiation. Initiation factors comparable to eIF1A, eIF2, eIF3, and f-Met-tRNA_i^Met then associate with the small subunit, followed by association of the large subunit to form the complete bacterial ribosome.

193