Because eIF2 binds very tightly to GDP, a guanine nucleotide exchange factor (see p. 157), designated eIF2B, is required to cause GDP release so that a new GTP molecule can bind and eIF2 can be reused (Figure 7–67A). The reuse of eIF2 is inhibited when it is phosphorylated—the phosphorylated eIF2 binds to eIF2B unusually tightly, inactivating eIF2B. There is more eIF2 than eIF2B in cells, and even a fraction of phosphorylated eIF2 can trap nearly all of the eIF2B. This prevents the reuse of the nonphosphorylated eIF2 and greatly slows protein synthesis (Figure 7–67B).
Regulation of the level of active eIF2 is especially important in mammalian cells; eIF2 is part of the mechanism that allows cells to enter a nonproliferating, resting state (called G0) in which the rate of total protein synthesis is reduced to about one-fifth the rate in proliferating cells.
initiation at AUG Codons Upstream of the translation Start Can regulate eukaryotic translation initiation
We saw in Chapter 6 that eukaryotic translation typically begins at the first AUG downstream of the 5ʹend of the mRNA, which is the first AUG encountered by a scanning small ribosomal subunit. But the nucleotides immediately surrounding the AUG also influence the efficiency of translation initiation. If the recognition site is poor enough, scanning ribosomal subunits will sometimes ignore the first AUG codon in the mRNA and skip to the second or third AUG codon instead. This phenomenon, known as “leaky scanning,” is a strategy frequently used to produce two or more closely related proteins, differing only in their N-termini, from the same mRNA. A particularly important use of this mechanism is the production of the same protein with and without a signal sequence attached at its N-terminus. This allows the protein to be directed to two different locations in the cell (for example, to both mitochondria and the cytosol). Cells can regulate the relative abundance of the protein isoforms produced by leaky scanning; for example, a cell-type-specific increase in the abundance of the initiation factor eIF4F favors the use of the AUG closest to the 5ʹ end of the mRNA.
Another type of control found in eukaryotes uses one or more short open reading frames—short stretches of DNA that begin with a start codon (ATG) and end with a stop codon, with no stop codons in between—that lie between the 5ʹend of the mRNA and the beginning of the gene. Often, the amino acid sequences coded by these upstream open reading frames (uORFs) are not important; rather, the uORFs serve a purely regulatory function. An uORF present on an mRNA molecule will generally decrease translation of the downstream gene by trapping a guanine nucleotide exchange factor, eIF2B
IN ABSENCE OF ACTIVE eIF2B, THE REMAINING UNBOUND eIF2
REMAINS IN ITS Figure 7–67 The eIF2 cycle. (A) the INACTIVE, GDP-recycling of used eif2 by a guanine BOUND FORM nucleotide exchange factor (eif2B). (B) eif2 (B) INACTIVE COMPLEX rates by tying up eif2B.
scanning ribosome initiation complex and causing the ribosome to translate the uORF and dissociate from the mRNA before it reaches the bona fide protein-coding sequence.