Activation and repression of translation

In Regulation of Gene Expression 1.  Operon Circuits in Bacteria and other Prokaryotes, we described mechanisms of repression as well as activation of translation in prokaryotes. Several proteins involved in repression and activation of translation are thus known in prokaryotes. However, in eukaryotes, no activator protein is known, and there is only one documented example of a translational repressor protein (iron-responsive element-binding protein = IRE-BP). On the other hand, regulation of translation in eukaryotes is also brought about by modification of general components (e.g. eIF2) of translation initiation machinery, a phenomenon unknown in prokaryotes. Regulation is also exercised during elongation of polypeptide chain, although its mechanism is not well understood.
Hypothetical mechanism for activation of translation in mammalian systems, (a) In the absence of activator, 5' end acquires a secondary structure, which does not allow binding of 40S; (b) binding of activator (triangle) opens the 5' end, by making an alternative stem-loop structure; (c) the activator is displaced by processivity of scanning 40S subunit of ribosome
Fig. 37.10. Hypothetical mechanism for activation of translation in mammalian systems, (a) In the absence of activator, 5' end acquires a secondary structure, which does not allow binding of 40S; (b) binding of activator (triangle) opens the 5' end, by making an alternative stem-loop structure; (c) the activator is displaced by processivity of scanning 40S subunit of ribosome.
Activator proteins. In eukaryotes, upto 1992, no unambiguous examples were available, where a m-RNA binding protein directly activates translation. However, one possible mechanism that may be involved in the action of an activation protein is shown in Figure 37.10. In some cases, the non-coding leader sequence at the 5' end forms a secondary structure, so that the 5' end is not available for binding of 40S subunit of ribosome. The activator protein binds to mRNA and leads to the formation of a hairpin structure, which is accompanied with the exposure of 5' end of mRNA for ribosome binding. The activator protein is later displaced by the scanning 40S subunit of ribosome, till it comes across the initiating codon AUG.
Hypothetical mechanism for activation of translation in mammalian systems, (a) In the absence of activator, 5' end acquires a secondary structure, which does not allow binding of 40S; (b) binding of activator (triangle) opens the 5' end, by making an alternative stem-loop structure; (c) the activator is displaced by processivity of scanning 40S subunit of ribosome
Fig. 37.10. Hypothetical mechanism for activation of translation in mammalian systems, (a) In the absence of activator, 5' end acquires a secondary structure, which does not allow binding of 40S; (b) binding of activator (triangle) opens the 5' end, by making an alternative stem-loop structure; (c) the activator is displaced by processivity of scanning 40S subunit of ribosome.

For transcription, many gene specific initiation factors (transcription factors) are known. For translation also, general initiation factors are known both in prokaryotes and eukaryotes, but no gene specific or m-RNA specific translation initiation factors have yet been identified in eukaryotes (although for translation of bacterial mRNA and mt mRNA, such factors are known).

Post-transcriptional regulation of two genes involved in the uptake and detoxification of iron in mammalian cells : (a) translation of mRNA for ferritin, a protein that sequesters excess iron, is inhibited by the binding of IRE-BP to mRNA at its 5' end; (b) translation of transferrin receptor (TfR) involved in the binding of IRE-BP to TfR mRNA at its 3' end; (c) actual sequence of one IRE (TfR mRNA has five such IRE motifs at 3' end; only one is shown here); (d) consensus IRE structure
Fig. 37.11. Post-transcriptional regulation of two genes involved in the uptake and detoxification of iron in mammalian cells : (a) translation of mRNA for ferritin, a protein that sequesters excess iron, is inhibited by the binding of IRE-BP to mRNA at its 5' end; (b) translation of transferrin receptor (TfR) involved in the binding of IRE-BP to TfR mRNA at its 3' end; (c) actual sequence of one IRE (TfR mRNA has five such IRE motifs at 3' end; only one is shown here); (d) consensus IRE structure. (See text for details).
Repressor protein (IRE-BP) : (a) Down-regulation of ferritin synthesis. The synthesis of ferritin (an iron-sequestering protein) in mammalian cells depends on iron availability. Its regulation is achieved through inhibition of the translation of ferritin mRNA, due to binding of a repressor protein at the 5' end of this mRNA. This 5' end contains the iron-response element (IRE), which makes a stem loop structure. The repressor protein is similarly called iron-response element binding protein (IRE-BP). IRE-BP resembles mitochondrial aconitase and has aconitase activity, enabling it to sense iron levels. When iron is scarce, the reduced form (3Fe-4S) of the protein binds the IRE and when iron is abundant, the oxidized form (4Fe-4S) dissociates from IRE. The stem-loop structure of IRE, located +10 to +30 nucleotides from 5' end of mRNA, allows binding of IRE-BP, inhibiting the initial binding of 40S subunit of ribosome. This reduces or stops the synthesis of ferritin from its mRNA (Fig. 37.11).
Post-transcriptional regulation of two genes involved in the uptake and detoxification of iron in mammalian cells : (a) translation of mRNA for ferritin, a protein that sequesters excess iron, is inhibited by the binding of IRE-BP to mRNA at its 5' end; (b) translation of transferrin receptor (TfR) involved in the binding of IRE-BP to TfR mRNA at its 3' end; (c) actual sequence of one IRE (TfR mRNA has five such IRE motifs at 3' end; only one is shown here); (d) consensus IRE structure
Fig. 37.11. Post-transcriptional regulation of two genes involved in the uptake and detoxification of iron in mammalian cells : (a) translation of mRNA for ferritin, a protein that sequesters excess iron, is inhibited by the binding of IRE-BP to mRNA at its 5' end; (b) translation of transferrin receptor (TfR) involved in the binding of IRE-BP to TfR mRNA at its 3' end; (c) actual sequence of one IRE (TfR mRNA has five such IRE motifs at 3' end; only one is shown here); (d) consensus IRE structure. (See text for details).

(b) Up-regulation of transferrin receptor (TfR) synthesis. The IRE-BP, while represses the synthesis of ferritin, it up-regulates the synthesis of transferrin receptor (TfR). The 3' end of TfR mRNA is prone to cleavage and IRE-BP binds there giving stability to TfR mRNA, so that it can be efficiently translated into TfR (Fig. 37.11).

IRE-BP also regulates translation of the first enzyme of heme biosynthetic pathway meant for iron utilization. Thus a single represser protein (IRE-BP) regulates both iron homeostasis and a metabolic pathway for iron utilization.

The eIF-2 cycle in mammalian cells. The elF-GDP complex released from the 40S ribosomal subunit in step(a) is stable. The exchange of GDP for GTP in step (b) is mediated by a protein, designated GEF, which is usually limiting in cells. Because GEF has a much higher affinity for eIF-2(αP) than for unmodified elf-2, phosphorylation of elF-2 sequesters GEF in a stable complex (step c), thus causing the global translational capacity to decline. The modification of eIF-2 and consequent inhibition of translation are reversed by phosphatases which are not depicted. The enzymes that catalyze phosphorylation of eIF-2 include p68-kinase (DAI) and the heme- regulated kinase HRI. The increase in phosphorylation depicted in step (c) is itself subject to down-regulation, for example, by VA-RNA of adenovirus. The other viruses marked with an asterisk also appear to encode or induce components that circumvent the virus-induced phosphorylation of eIF-2
Fig. 37.12. The eIF-2 cycle in mammalian cells. The elF-GDP complex released from the 40S ribosomal subunit in step(a) is stable. The exchange of GDP for GTP in step (b) is mediated by a protein, designated GEF, which is usually limiting in cells. Because GEF has a much higher affinity for eIF-2(αP) than for unmodified elf-2, phosphorylation of elF-2 sequesters GEF in a stable complex (step c), thus causing the global translational capacity to decline. The modification of eIF-2 and consequent inhibition of translation are reversed by phosphatases which are not depicted. The enzymes that catalyze phosphorylation of eIF-2 include p68-kinase (DAI) and the heme- regulated kinase HRI. The increase in phosphorylation depicted in step (c) is itself subject to down-regulation, for example, by VA-RNA of adenovirus. The other viruses marked with an asterisk also appear to encode or induce components that circumvent the virus-induced phosphorylation of eIF-2 (see text).
Translational control by phosphorylation of the components of translation machinery Reversible phosphorylation of eIF-2. In mammalian systems, reversible phosphorylation of eIF-2 has been known for years, although its role in regulation of translation has been proved only recently. As shown in Expression of Gene : Protein Synthesis 4. Translation in Prokaryotes and Eukaryotes, eIF-2 forms a complex with GTP, which binds with met-tRNAfet and carries it onto the 40S ribosome subunit, forming a 43S complex. The 43S complex engages at the 5' end of mRNA and scans down to the AUG codon, where eIF-5 joins and causes hydrolysis of GTP. The resulting eIF-2 • GDP has lower affinity for 40S and is therefore released, leaving 40S met-tRNAfe' behind. The 60S then joins giving 80S ribosome, ready to form the first peptide bond (Fig. 37.12).
The eIF-2 cycle in mammalian cells. The elF-GDP complex released from the 40S ribosomal subunit in step(a) is stable. The exchange of GDP for GTP in step (b) is mediated by a protein, designated GEF, which is usually limiting in cells. Because GEF has a much higher affinity for eIF-2(αP) than for unmodified elf-2, phosphorylation of elF-2 sequesters GEF in a stable complex (step c), thus causing the global translational capacity to decline. The modification of eIF-2 and consequent inhibition of translation are reversed by phosphatases which are not depicted. The enzymes that catalyze phosphorylation of eIF-2 include p68-kinase (DAI) and the heme- regulated kinase HRI. The increase in phosphorylation depicted in step (c) is itself subject to down-regulation, for example, by VA-RNA of adenovirus. The other viruses marked with an asterisk also appear to encode or induce components that circumvent the virus-induced phosphorylation of eIF-2
Fig. 37.12. The eIF-2 cycle in mammalian cells. The elF-GDP complex released from the 40S ribosomal subunit in step(a) is stable. The exchange of GDP for GTP in step (b) is mediated by a protein, designated GEF, which is usually limiting in cells. Because GEF has a much higher affinity for eIF-2(αP) than for unmodified elf-2, phosphorylation of elF-2 sequesters GEF in a stable complex (step c), thus causing the global translational capacity to decline. The modification of eIF-2 and consequent inhibition of translation are reversed by phosphatases which are not depicted. The enzymes that catalyze phosphorylation of eIF-2 include p68-kinase (DAI) and the heme- regulated kinase HRI. The increase in phosphorylation depicted in step (c) is itself subject to down-regulation, for example, by VA-RNA of adenovirus. The other viruses marked with an asterisk also appear to encode or induce components that circumvent the virus-induced phosphorylation of eIF-2 (see text).

Affinity of eIF-2 for GDP is 400-fold higher than for GTP and therefore regeneration of eIF-2 GTP from eIF-2 • GDP requires a helper factor, designated eIF-2B or GEF (guanine exchange factor). However, the regeneration of eIF-2 GTP may be prevented, if α subunit of elF- 2 undergoes phosphorylation, which is promoted by a variety of conditions including heat shock, amino acid deprivation or virus infection. GEF has a 150-fold higher affinity for eIF-2 (αP) than for unmodified elF- 2, so that formation of stable eIF-2 (αp) • GDP. GEF does not allow the formation of eIF-2 • GTP, (due to depletion of GEF) causing protein synthesis (translation) to stop (Fig.37.12).AnexampleofelF-2 mediated regulation of the translation of mRNA for the yeast gene GCN4 is discussed below.

(a) Translational control of the gene GCN4 in yeast via eIF-2. In yeast, expression of GCN4 gene (involved in general control of nitrogen metabolism) is regulated primarily at translation level, since although under different conditions, the level of protein encoded by GCN4 changes more than 100 fold, the level of mRNA for this gene does not change. GCN4 gene protein (a transcription factor) regulates transcription of 30-40 genes involved in amino acid biosynthesis and its own synthesis is boosted 100 fold when yeast is starved of an essential amino acid. Its level of expression also depends on a repressor specified by GCD1 locus, which in its turn is regulated by GCN1, 2 and 3 loci.

Structures of mRNAs corresponding to genes for GCN4 (general control of nitrogen metabolism) and ornithine decarboxylase in yeast, showing untranslated sequence at 5' end having false AUG codons
Fig. 37.13. Structures of mRNAs corresponding to genes for GCN4 (general control of nitrogen metabolism) and ornithine decarboxylase in yeast, showing untranslated sequence at 5' end having false AUG codons.
The structure of mRNA of GCN4 gene explains the mechanism of its translational regulation, on the basis of Kozak's scanning hypothesis discussed in Expression of Gene : Protein Synthesis 4. Translation in Prokaryotes and Eukaryotes. It has a 577 nucleotides long untranslated sequence at 5' end, which is much longer than normal and has four AUG .codons upstream of the real AUG (Fig. 37.13). The presence of four AUG codons is utilized by the eukaryotic initiation factor, eIF2 in the regulation of translation of GC7V4-mRNA. The initiation factor, eIF-2 forms a complex eIF-2 GTP-met-tRNA, whose concentration is regulated by amino acid concentration.
Under amino acid starvation, uncharged tRNAmet accumulates and activates a protein kinase, which phosphorylates and inactivates a portion of eIF-2 molecules, thus reducing the concentration of eIF-2 in the cell. Since eIF-2 escorts met-tRNAmet onto ribosomes, under reduced concentration of eIF-2, 40S subunit will require more time to acquire met-tRNAmet and become competent for reinitiation.
Structures of mRNAs corresponding to genes for GCN4 (general control of nitrogen metabolism) and ornithine decarboxylase in yeast, showing untranslated sequence at 5' end having false AUG codons
Fig. 37.13. Structures of mRNAs corresponding to genes for GCN4 (general control of nitrogen metabolism) and ornithine decarboxylase in yeast, showing untranslated sequence at 5' end having false AUG codons.

Translation control of GCN4 gene in yeast through reinitiation mechanism; only two of the four AUG codons (shown as 1 and 4) are depicted (see text for details)
Fig 37.14. Translation control of GCN4 gene in yeast through reinitiation mechanism; only two of the four AUG codons (shown as 1 and 4) are depicted (see text for details).
Due to slower acquisition of competence, some 40S subunits bypass one or more AUG codons and gain access to the GCN4 start site. On the other hand, under condition of no starvation (amino acid), eIF-2 concentration is adequate so that the 40S subuims become competent quickly and can not bypass the fourth AUG codon. Consequently, a small peptide is synthesized and released at the termination site of ORF4 (open reading frame-4).
The termination at 0RF4 is somehow incompatible with reinitiation at GCN4, so that under condition of no starvation, GCN4 is not expressed. Translation of GCN4-mRNA is also inhibited, if intercistronic distance between ORF1 and ORF4 is increased by inserting an unknown sequence. In this case, a longer distance allows all 40S subunits become competent before reaching 0RF4, even under starvation condition. This will not leave any 40S subunit to bypass ORF4 and reinitiate translation at GCN4. (Fig. 37.14).
Translation control of GCN4 gene in yeast through reinitiation mechanism; only two of the four AUG codons (shown as 1 and 4) are depicted (see text for details)
Fig 37.14. Translation control of GCN4 gene in yeast through reinitiation mechanism; only two of the four AUG codons (shown as 1 and 4) are depicted (see text for details).

(b) Translational control of the synthesis of ornithine decarboxylase. An example analogous to GCN4 gene in yeast, is the mRNA corresponding to ornithine decarboxylase enzyme involved in polyamine biosynthesis. It contains a 737 nucleotides long 5' untranslated region containing four AUG codons (Fig. 37.13) followed by termination codons 16, 2, 4 and 10 nucleotides later respectively. The enzyme is extremely rapidly produced in cells stimulated by hormones and growth factors, but we do not know if translational efficiency changes with the physiological state of the cell. If so, the long leader sequence and the false AUG codons may play a part.

Reversible phosphorylation of eIF-4E and rpS6 (ribosomal protein S6). Besides eIF-2, eIF-4E and rpS6 have also been observed to undergo striking changes in the level of phosphorylation under specific conditions like serum deprivation in culture medium (eIF-4E) and heat shock. Under heat shock, phosphorylation of eIF-2 increases and that of rpS6 decreases. Also, while increased phosphorylation of eIF-2 leads to reduced translation, increased phosphorylation of rpS6 apparently leads to enhanced translation.

In case of eIF-4E, the principal site of phosphorylation is 'serine-53' and a mutation on this site renders the factor non-functional in translation assays. This suggests that eIF-4E may undergo reversible phosphorylation each time it is used. In activated B lymphocytes also, increased phosphorylation was associated with increased translation rate.

One of the cell division cycle (cdc)genes in yeast also encodes eIF-4E, which may regulate cell division by reversible phosphorylation. Phosphorylation of eIF-2, eIF-4E and rpS6 is also associated with phosphorylation of other initiation factors, elongation factors and aminoacyl tRNA synthetases. However, unequivocal evidence of the regulation of translation by reversible phosphorylation is available only in case of eIF-2.

Reversible phosphorylation and targeting specific mRNA. The regulation at the translation level requires that the phosphorylated or dephosphorylated forms of the components of translational machinery, should identify a specific mRNA from the bulk mRNA population. This may be achieved in one of the following ways : (i) a mechanism like that for GCN4 described above (several AUG codons upstream of real start codon); (ii) activation of kinases or phosphatases in a localized region of cytosol, where a specific mRNA is localized (compartmentalization of the cytosol); (iii) variation in secondary and tertiary structures among the non-coding leader sequences at the 5' ends of mRNA molecules, which modulate ribosome-mRNA interaction.

The best example of differential translation of mRNAs is the virus infected cells, where viral genes are preferentially expressed. This may be achieved by one of the following mechanisms : (i) Cellular mRNAs are degraded or host's transcription is shut off. (ii) Viral mRNAs are more abundant (e.g. reovirus). (iii) Viral mRNAs are better designed for initiating translation, due to unstructured 5' leader sequence, (e.g. adenovirus). (iv) Overall translational capacity is reduced due to partial phosphorylation of eIF-2a, induced by double stranded RNA generated during the course of virus transcription and/or translation (e.g. adenovirus, poliovirus, reovirus, etc.). In many viruses, factors are also generated which inhibit completion of phosphorylation of eIF-2α. An adenovirus encoded transcript, designated VA­RNA, actually binds to and blocks the activation of p68 kinase, thus making unaltered eIF-2α efficient for translation.

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