Initiation Involves Several ProteinRNA Complexes Figure 386

Initiation of protein synthesis requires that an mRNA molecule be selected for translation by a ribosome. Once the mRNA binds to the ribosome, the latter finds the correct reading frame on the mRNA, and translation begins. This process involves tRNA, rRNA, mRNA, and at least ten eukaryotic initiation factors (elFs), some of which have multiple (three to eight) subunits. Also involved are GTP, ATP, and amino acids. Initiation can be divided into four steps: (1) dissociation of the ribosome into its 40S and 60S sub-units; (2) binding of a ternary complex consisting of met-tRNA1, GTP, and eIF-2 to the 40S ribosome to form a preinitiation complex; (3) binding of mRNA to the 40S preinitiation complex to form a 43S initiation complex; and (4) combination of the 43S initiation complex with the 60S ribosomal subunit to form the 80S initiation complex.

A. Ribosomal Dissociation_

Two initiation factors, eIF-3 and eIF-1A, bind to the newly dissociated 40S ribosomal subunit. This delays its reassociation with the 60S subunit and allows other translation initiation factors to associate with the 40S subunit.

B. Formation of the 43S Preinitiation Complex

The first step in this process involves the binding of GTP by eIF-2. This binary complex then binds to met-tRNAi, a tRNA specifically involved in binding to the initiation codon AUG. (There are two tRNAs for me-thionine. One specifies methionine for the initiator codon, the other for internal methionines. Each has a unique nucleotide sequence.) This ternary complex binds to the 40S ribosomal subunit to form the 43S preinitiation complex, which is stabilized by association with eIF-3 and eIF-1A.

eIF-2 is one of two control points for protein synthesis initiation in eukaryotic cells. eIF-2 consists of a, P, and Y subunits. eIF-2a is phosphorylated (on serine 51) by at least four different protein kinases (HCR, PKR, PERK, and GCN2) that are activated when a cell is under stress and when the energy expenditure required for protein synthesis would be deleterious. Such conditions include amino acid and glucose starvation, virus infection, misfolded proteins, serum deprivation, hyperosmolality, and heat shock. PKR is particularly interesting in this regard. This kinase is activated by viruses and provides a host defense mechanism that decreases protein synthesis, thereby inhibiting viral replication. Phosphorylated eIF-2a binds tightly to and inactivates the GTP-GDP recycling protein eIF-2B. This prevents formation of the 43S preini-tiation complex and blocks protein synthesis.

C. Formation of the 43S Initiation Complex

The 5' terminals of most mRNA molecules in eukaryotic cells are "capped," as described in Chapter 37. This methyl-guanosyl triphosphate cap facilitates the binding of mRNA to the 43S preinitiation complex. A cap binding protein complex, eIF-4F (4F), which consists of eIF-4E and the eIF-4G (4G)-eIF4A (4A) complex, binds to the cap through the 4E protein. Then eIF-4A (4A) and eIF-4B (4B) bind and reduce the complex secondary structure of the 5' end of the mRNA through ATPase and ATP-dependent helicase activities. The association of mRNA with the 43S preinitiation complex to form the 48S initiation complex requires ATP hydrolysis. eIF-3 is a key protein because it binds with high affinity to the 4G component of 4F, and it links this complex to the 40S ribosomal subunit. Following association of the 43S preinitiation complex with the mRNA cap and reduction ("melting") of the secondary structure near the 5' end of the mRNA, the complex scans the mRNA for a suitable initiation codon. Generally this is the 5'-most AUG, but the precise initiation codon is determined by so-called Kozak consensus sequences that surround the AUG:

GCCA/GCCAUGG

Most preferred is the presence of a purine at positions -3 and +4 relative to the AUG.

D. Role of the Poly(A) Tail in Initiation_

Biochemical and genetic experiments in yeast have revealed that the 3' poly(A) tail and its binding protein, Pab1p, are required for efficient initiation of protein synthesis. Further studies showed that the poly(A) tail stimulates recruitment of the 40S ribosomal subunit to the mRNA through a complex set of interactions. Pab1p, bound to the poly(A) tail, interacts with eIF-4G, which in turn binds to eIF-4E that is bound to the cap structure. It is possible that a circular structure is formed and that this helps direct the 40S ribosomal subunit to the 5' end of the mRNA. This helps explain how the cap and poly(A) tail structures have a synergis-tic effect on protein synthesis. It appears that a similar mechanism is at work in mammalian cells.

Ternary complex formation

Formation of the 80S initiation complex

Activation of mRNA

GDPO

Formation of the 80S initiation complex

Activation of mRNA

GDPO

Met eIF-5

Met eIF-5

P site

Elongation

Figure 38-6. Diagrammatic representation of the initiation of protein synthesis on the mRNA template containing a 5' cap (GmTP-5') and 3' poly(A) terminal [3'(A)n]. This process proceeds in three steps: (1) activation of mRNA; (2) formation of the ternary complex consisting of tRNAmet1, initiation factor eIF-2, and GTP; and (3) formation of the active 80S initiation complex. (See text for details.) GTP, •; GDP, o. The various initiation factors appear in abbreviated form as circles or squares, eg, eIF-3 (O), eIF-4F (I4F I). 4«F is a complex consisting of 4E and 4A bound to 4G (see Figure 38-7). The constellation of protein factors and the 40S ribosomal subunit comprise the 43S preinitiation complex. When bound to mRNA, this forms the 48S preinitiation complex.

P site

E. Formation of the 80S Initiation Complex

The binding of the 60S ribosomal subunit to the 48S initiation complex involves hydrolysis of the GTP bound to eIF-2 by eIF-5. This reaction results in release of the initiation factors bound to the 48S initiation complex (these factors then are recycled) and the rapid association of the 40S and 60S subunits to form the 80S ribosome. At this point, the met-tRNA1 is on the P site of the ribosome, ready for the elongation cycle to commence.

Diabetes 2

Diabetes 2

Diabetes is a disease that affects the way your body uses food. Normally, your body converts sugars, starches and other foods into a form of sugar called glucose. Your body uses glucose for fuel. The cells receive the glucose through the bloodstream. They then use insulin a hormone made by the pancreas to absorb the glucose, convert it into energy, and either use it or store it for later use. Learn more...

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