Translational &Transcriptional Control Of Gene Expressi

Child Health And Human Development

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Abstract

We study a global regulatory mechanism in yeast that mediates transcriptional induction of genes encoding amino acid biosynthetic enzymes in response to starvation for any amino acid. The transcriptional activator in this pathway, GCN4, is induced at the translational level in amino acid starved cells by phosphorylation of translation initiation factor 2 (eIF2) on its alpha subunit by protein kinase (PK) GCN2. To identify all of the genes in yeast that are transcriptionally induced by GCN4, we conducted mRNA expression profiling using cDNA microarrays. Remarkably, we found that ~1000 genes were induced and ~1000 genes were repressed two-fold or more in response to histidine starvation. Profiling of a gcn4 deletion mutant showed that the majority of these genes were dependent on GCN4 for their induction or repression upon histidine starvation. Interestingly, we found that a subset of GCN4 target genes is subject to dual regulation by GCN4-dependent and GCN4-independent regulatory mechanisms. Repression by GCN4 was found to be indirect and may involve squelching of transcription factors that are downregulated in starved cells. In addition to inducing nearly all genes encoding amino acid biosynthetic enzymes, GCN4 induced genes involved in the biosynthesis of amino acid precursors, purines, and nearly all of the vitamins. GCN4 also induced peroxisomal genes and mitochondrial transporter proteins, likely reflecting the occurrence of amino acid biosynthetic steps in these organelles. Autophagy genes and amino acid permeases were induced, presumably to boost amino acid pools by nonbiosynthetic routes. Several hundred additional genes without obvious biosynthetic functions were induced, including numerous transcriptional activators, protein kinases and protein phosphatases. Thus, GCN4 seems to mobilize multiple regulatory circuits in addition to boosting the biosynthetic capacity of the cell in response to nutrient starvation. Protein kinase GCN2 is activated in amino acid-starved cells by binding of uncharged tRNA to a histidyl-tRNA synthetase (HisRS)-related region adjacent to the PK domain, that functions as a general sensor of amino acid limitation. The C-terminal domain (Cterm) of GCN2 interacts with the PK domain and also is required for tRNA binding, leading us to propose that tRNA binding eliminates an autoinhibitory interaction between the PK domain and Cterm. Recent findings indicate that the N-terminal portion of the HisRS region (HisRS-N) also interacts with the PK domain and stimulates kinase activity. This implies that the HisRS-N and PK domains are engaged in the tRNA-bound, activated state. Point mutations in the PK domain have been isolated that activate kinase function independently of tRNA binding. These mutations likely remove an inhibitory structure in the kinase domain that is normally relieved by interaction with the HisRS-N domain bound to tRNA. GCN1 and GCN20 are positive effectors of GCN2, both related to translation elongation factor EF3, that reside in a protein complex with ribosome-binding activity. GCN1 binds directly to GCN2 and discrete segments in each protein required for the interaction have been identified. Genetic analysis confirms that GCN1-GCN2 association is crucial for GCN2 activation in starved cells. GCN1 binding to ribosomes increases sensitivity to paromomycin, an inhibitor of the decoding (A) site on the ribosome. We propose that ribosome-bound GCN1/GCN20 complexes bind to GCN2 and facilitate transfer of uncharged tRNA from the A site to the HisRS domain in GCN2. The human kinase PKR is activated by double-stranded RNA (dsRNA) and phosphorylates the alpha subunit of eIF2 to inhibit protein synthesis in virus-infected cells. PKR contains two dsRNA binding motifs (DRBMs I and II) required for its activation by dsRNA. By progressively removing dsRNA contacts in the DRBMs with alanine substitutions, we found that dimerization of PKR expressed in yeast cells was impaired by the minimal combinations of mutations that impaired dsRNA binding in vitro. Human PKR contains at least 15 autophosphorylation sites, but only T446 and T451 in the PK activation loop were critically required for kinase activity in yeast cells. Using a phosphospecific antibody, we showed that T451 phosphorylation is stimulated by dsRNA binding. Our results provide strong evidence that dsRNA binding is required for dimerization of PKR molecules, leading to autophosphorylation of the activation loop and activation of its eIF2 kinase function. Phosphorylation of the alpha subunit of eIF2 on Serine-51 inhibits the 5-subunit guanine nucleotide exchange factor for eIF2, known as eIF2B. This prevents formation of the ternary complex (TC) containing eIF2, GTP, and initiator methionyl tRNA that transfers tRNAiMet to the 40S ribosome. Three of the eIF2B subunits in yeast (GCN3, GCD7, and GCD2) comprise a regulatory subcomplex that binds phosphorylated eIF2 (P-eIF2) and mediates inhibition of the GCD1/GCD6 catalytic subcomplex of eIF2B. We have shown that the alpha subunit of eIF2 (SUI2) alone can bind directly to the eIF2B regulatory subcomplex, dependent on Ser-51 phosphorylation. Mutations in SUI2 residues surrounding Ser-51, or in the GCD7 subunit, that abolish regulation of eIF2B by P-eIF2 in vivo impaired binding of eIF2B to P-SUI2 in vitro. Thus, tight binding of P-SUI2 to the eIF2B regulatory subcomplex is required for the inhibition of eIF2B by P-eIF2. Human initiation factor 3 (eIF3) is a 10-subunit complex that stimulates binding of the TC and mRNA to the 40S ribosome in vitro. Yeast eIF3 contains 5 core subunits and a sixth more loosely associated protein known as HCR1. Protein interaction assays have led to a detailed subunit interaction map for eIF3 and also shown that eIF3c/NIP1 provides a binding site for eIFs 1 and 5 in the eIF3 complex. eIF5, the GTPase activating protein for the TC, and eIF1, were both implicated previously in selection of AUG as the start codon. This common function may require their mutual interaction with the N-terminus of NIP1. A stable eIF3 subcomplex containing only three of the core eIF3 subunits, eIF3a/TIF32, eIF3b/PRT1 and eIF3c/NIP1 was overexpressed in yeast cells and shown to associate with HCR1, eIF1 and eIF5. This purified subcomplex rescued initiator tRNA- and mRNA-binding in heat-inactivated extracts of a prt1 mutant, whereas a stable eIF3b/g/i subcomplex was nearly inactive in these assays. Thus, the three largest eIF3 subunits are sufficient for its critical functions in initiation complex assembly. The C-terminal segment of eIF5 (CTD), which binds to NIP1, also interacts with the beta subunit of eIF2, and these interactions can occur simultaneously in vitro. A multifactor complex (MFC) containing eIFs 1,2,3, 5 and tRNAiMet was detected in vivo free of ribosomes. An eIF5 mutation that disrupted these interactions (tif5-7A) impaired translation in vivo, suggesting that the MFC is an important intermediate in translation initiation. The tif5-7A mutation impairs TC and mRNA binding to 40S ribosomes in vitro, consistent with a simulatory role for the eIF5-CTD in recruitment of TC and mRNA to the 48S initiation complex. In vivo, however, the rate-limiting defect in the tif5-7A mutant involves loss of eIF5 as a stable constituent of the 48S complex, leading to a defect in AUG recognition or GTP hydrolysis in the TC. Thus eIF5 must enter the 48S complex in tight association with the TC and eIF3 to execute its function as the GAP for the TC on AUG recognition.