13.5: Eukaryotic Translation Regulation

mRNAs are made to be translated, and so they are! Nonetheless, translation is regulated, largely by controlling translation initiation. Let’s be reminded about the basics of eukaryotic initiation. In many respects, the overall process is similar to prokaryotic translation initiation, described elsewhere. The 40S ribosomal subunit itself can bind to and scan an mRNA, seeking the start site of an ORF (open reading frame) encoding a polypeptide. When a GTP-bound eukaryotic initiation factor 2 (GTP-eIF2) binds with met-tRNAf, it forms a ternary complex (TC). The TC can associate with the scanning 40S subunit. When a TC-associated scanning subunit encounters the start site of the ORF, scanning stalls. Additional eukaryotic initiation factors (eIFs) help form the initiation complex, positioning the initiator tRNA anticodon over the start site AUG in the mRNA. The initiation complex then recruits the large (60S) ribosomal subunit. Since most of the regulation of translation involves controlling the activity of initiation factors and the structure of the promoter region of genes, watch for these interactions in the following descriptions. Let’s begin with a close look at Look at translation initiation in eukaryotes (Figure 13.9). Recall that the binding of the 60S ribosomal subunit to the initiation complex (lower left) causes the release of all the eIFs and the hydrolysis of the GTP on eIF2; GDP remains bound to eIF2 (upper right).

Figure 13.9: Review of the steps in Eukaryotic translation initiation.

For protein syntheses to continue, new GTP must replace GDP on eIF2. Another initiation factor, eIF2B, facilitates this GTP/GDP swap, recycling GTP-eIF2 for use in initiation (i.e., the association of eIF2, the initiator tRNAf and formyl methionine to form the TC). The regulation of translation is superimposed on these basic processes. But where? What steps in initiation are controlled?

We know that the 5’ methyl-guanosine caps and poly(A) tails on mRNAs are required for efficient translation, because mRNAs engineered to lack one and/or the other are poorly translated. But there is little evidence that cells control capping, polyadenylation, or the structures themselves. Instead, translation is regulated largely by targeting interactions with structural features and sequences in the 5’ region of mRNAs. Figure 13.10 below shows key structural motifs in a gene that can contribute to translational control.

Figure 13.10: Regions and specific sequences known to be involved in regulating translation.

Regulation of translation may be global, affecting the synthesis of many polypeptides; or it may be specific, affecting a single polypeptide. The global regulation involves changes in the activity of eIFs that would typically affect all cellular protein synthesis. Specific regulation involves sequences or regions on one or a few mRNAs in turn can recognize and bind specific regulatory proteins and/or other molecules. Such specific interactions would control translation of only those mRNAs, without affecting overall protein biosynthesis. We will consider three examples of the translational control of gene expression.

13.5.1. Specific Translation Control by mRNA-Binding Proteins

Translation initiation complexes typically scan the 5’ UTR of an mRNA. When the complex finds the translation start site, it can bind the large subunit and begin translating the polypeptide. Ferritin is a cytplasmic iron-binding protein, necessary because iron is of course, insoluble and must be bound to proteins, both in cells and in the blood (the latter is a story for another time and place!). Once in the cell, iron binds to the ferritin. As you may guess, cellular iron metabolism depends on the amount of ferritin in the cell, which is regulated at the level of translation. Here we consider the control of ferritin synthesis (Figure 13.11, below).

Ferritin is in fact a two-subunit protein. The 5’ UTRs of the mRNAs for both polypeptide chains contain iron-responsive elements (IREs). These IREs are stem-loop structures that recognize and specifically bind iron-regulatory proteins (IRP1, IRP2). The initiation complex scans the 5’ UTR of an mRNA. When it finds the normal translationstart site, it can bind the large subunit and begin translating the polypeptide. In iron-deficient cells, scanning by the initiation complex is thought to be physically blocked by steric hindrance.

Figure 13.11: Iron-regulatory proteins disrupt promoter scanning to inhibit ferritin-polypeptide synthesis.

13.5.2 Coordinating Heme and Globin Synthesis

Reticulocytes (the precursors to erythrocytes, the red blood cells in mammals) synthesize globin proteins. They also synthesize heme, an iron-bound porphyrin-ring molecule. Each globin must bind to a single heme to make a hemoglobin protein subunit. Clearly, it would not do for a reticulocyte to make too much globin protein and not enough heme, or vice versa. In fact, hemin (a precursor to heme) regulates the translation initiation of both \(\alpha\) and \(\beta\) globin mRNAs. Recall that, in order to sustain globin mRNA translation—the GDP-eIF2 generated after each cycle of translation elongation must be exchanged for fresh GTP. This is facilitated by the eIF2B initiation factor. eIF2B can be phosphorylated (inactive) or unphosphorylated (active). Making sure that globin is not under- or overproduced relative to heme biosynthesis involves controlling levels of active vs inactive eIF2B by hemin. Hemin accumulates when there is not enough globin polypeptide to combine with heme in the cell.

Figure 13.12 below illustrates the regulation of globin mRNA translation initiation by hemin. Excess hemin binds and inactivates an HCR kinase, preventing the phosphorylation of eIF2B. Since unphosphorylated eiF2B is active, it facilitates the GTP/GDP swap needed to allow continued translation. Thus, ongoing initiation ensures that globin mRNA translation can keep up with heme levels. In other words, if hemin production gets ahead of globin, it will promote more globin translation until globin and heme levels are nearly equimolar. Since hemin is no longer in excess, it dissociates from the inactive HCR kinase. The now-active kinase catalyzes eIF2B phosphorylation. Now, phospho-eIF2B is inactive and can’t facilitate the GTP/GDP swap on eIF2. Globin mRNA translation initiation, now blocked, slows the rate of globin polypeptide translation to keep pace with heme synthesis.

Figure 13.12: Trace how hemin (a heme precursor) coordinates globin polypeptide translation with its own synthesis (clockwise in panels 1-4). The net results are that at high [hemin], globin polypeptide translation is maximal, while at low [hemin], globin translation slows down.

CHALLENGE

The regulation of globin translation seems to be specific. But wouldn’t changing translation-factor activity affect the translation of all proteins? What do you think is going on here?

237 Translation Regulation of Globin Polypeptide Synthesis

13.5.3 Translational Regulation of Yeast GCN4

Like the coordination of heme and globin production, regulation of the yeast GCN4 protein is based on controlling the ability of the cells to swap GTP for GDP on eIF2. But this regulation is quite a bit more complex even though yeast is a more primitive eukaryote! GCN4 is a global transcription factor, controlling the transcription of as many as thirty genes in pathways for the synthesis of nineteen out of the twenty amino acids! The discovery that amino acid starvation caused yeast cells to increase production of amino acids led to the discovery of the General Amino Acid Control (GAAC) mechanism involving GCN4. GCN is short for General Control Nondepressible, referring to its global, positive regulatory effects. It turns out that the GCN4 protein is also involved in stress-gene expression, glycogen homeostasis, purine biosynthesis… the list just goes on. In fact, the GCN4 protein is involved in the action of up to 10% of all yeast genes! Here we focus on the GAAC mechanism.

Yeast cells with ample amino acids don’t need to make more. GCN4 levels are low in these cells. But if the cells are starved of amino acids, GCN4 levels rise as much as tenfold in two hours, causing increased general amino acid synthesis. Amino acid starvation initially signals an increase in the activity of GCN2, another protein kinase that catalyzes GDP-eIF2 phosphorylation (Figure 13.13), which we just saw, cannot exchange GTP for GDP on eIF2.

Figure 13.13: Amino acid starvation increases a GCN2 kinase activity that phosphorylates GDP-eIF2 (1). eIF2B then binds GDP-eIF2-P (2), blocking GTP/GDP exchange (3). The resulting drop in ternary complexes (4) favors GCN4 translation, leading to maximal amino acid synthesis (5).

There is a paradox here. You would expect a slowdown in GTP-eIF2 regeneration to inhibit overall protein synthesis, and it does. However, the reduced levels of GTP-eIF2 somehow also stimulate translation of the GCN4 mRNA, leading to more transcription of the amino acid synthesis genes. In other words, amino acid starvation leads yeast cells to use available substrates to make their own amino acids, so that protein synthesis can continue… at the same time as initiation complex formation is disabled! Let’s accept that paradox for now and look at how amino acid starvation leads to increased translation of the GCN4 protein and the upregulation of amino acid biosynthesis pathways. To begin with, we are going to need to understand the structure of GCN4 mRNA (Figure 13.14). Note the four short upstream open reading frames (uORFs) in the 5’ untranslated region (5’ UTR) of GCN4 mRNA.

Figure 13.14: Short open reading frames (uORFs) in the GCN4 mRNA 5′ UTR act to regulate translation.

Recall that eIF2 binds with GTP and the initiator met-tRNA to form a ternary complex (TC). As we also noted earlier, TC-associated 40S ribosomal subunits scan mRNAs to find translation start sites of ORFs, allowing initiation complexes to form. When a 60S ribosomal subunit binds, polypeptide translation starts. But while uORFs in the GCN4 mRNA encode only a few amino acids before encountering a stop codon, they can also be recognized during scanning. When TCs and 40S subunits are plentiful, they engage uORFs in preference to the GCN4-coding region ORF (Figure 13.15).

Figure 13.15: At normal amino acid levels, abundant TC-40S (ternary) complexes scanning for ORFs cluster at uORF start sites, slowing scanning to the GCN4 ORF and thus inhibit GCN4 translation.

Under these conditions, active eIF2B allows the GTP/GDP swap on GDP-eIF2, leading to efficient GTP-eIF2 recycling and high TC levels. TCs were bound to the small (40S) subunits during scanning and/or at the start sites of uORFs. At this point, they form initiation complexes that bind 60S ribosomal subunits and begin uORF translation.

The effect of high TC levels, leads to high levels of initiation complexes that in turn drive scanning for the GCN4 ORF, slowing down scanning the upstream ORFs (uORFs). thereby inhibiting initiation complex formation at the actual GCN4 ORF. The result is increased transcription of GCN4 mRNA and GCN4 protein synthesis.

CHALLENGE

State an hypothesis to explain how higher TC levels result in more efficient scanning and thus initiation at the GCN4 promoter.

What happens in amino acid-starved cultures of yeast cells, when GTP-eIF2 cannot be efficiently regenerated and TCs are in short supply? To review, amino acid starvation signals an increase in GCN2 kinase activity, resulting in phosphorylation and inactivation of eIF2B. Inactive phospho-eIF2 will not facilitate the GTP/GDP swap at GDP-eIF2, inhibiting overall protein synthesis. The resulting reduction in GTP-eIF2 also lowers the levels of TC and TCassociated 40S subunits. In Figure 13.16 (below), see how this phenomenon upregulates GCN4 translation, even as the translation of other mRNAs has declined.

Figure 13.16: During amino acid starvation there are fewer TC-40S complexes scanning for ORFs and they more readily bind to the GCN4 ORF, increasing GCN4 synthesis. At high levels, GCN4 regulates the expression of many genes, including those encoding enzymes needed for amino acid synthesis.

Under these conditions, initiation complex formation at uORFs is less likely, and scanning by remaining TC-40S complexes is more likely to reach the GCN4 ORF and to increase GCN4 translation! The increased production of the gene-regulatory GCN4 protein then turns on virtually all of the genes for enzymes of amino acid biosynthesis. The cells thus make their own amino acids when the medium has few to contribute! For a good review of translation-level regulation in general and of GCN4, see GCN4 Translation Regulation.