Interaction between the tRNA-Binding and C-Terminal Domains of Yeast Gcn2 Regulates Kinase Activity In Vivo

Citation: Lageix, S., Zhang, J. W., Rothenburg, S., & Hinnebusch, A. G. (2015). Interaction between the tRNA-Binding and C-Terminal Domains of Yeast Gcn2 Regulates Kinase Activity In Vivo. Plos Genetics, 11(2), 28. doi:10.1371/journal.pgen.1004991The stress-activated protein kinase Gcn2 regulates protein synthesis by phosphorylation of translation initiation factor eIF2 alpha. Gcn2 is activated in amino acid-deprived cells by binding of uncharged tRNA to the regulatory domain related to histidyl-tRNA synthetase, but the molecular mechanism of activation is unclear. We used a genetic approach to identify a key regulatory surface in Gcn2 that is proximal to the predicted active site of the HisRS domain and likely remodeled by tRNA binding. Mutations leading to amino acid substitutions on this surface were identified that activate Gcn2 at low levels of tRNA binding (Gcd(-) phenotype), while other substitutions block kinase activation (Gcn(-) phenotype), in some cases without altering tRNA binding by Gcn2 in vitro. Remarkably, the Gcn(-) substitutions increase affinity of the HisRS domain for the C-terminal domain (CTD), previously implicated as a kinase autoinhibitory segment, in a manner dampened by HisRS domain Gcd(-) substitutions and by amino acid starvation in vivo. Moreover, tRNA specifically antagonizes HisRS/CTD association in vitro. These findings support a model wherein HisRS-CTD interaction facilitates the autoinhibitory function of the CTD in nonstarvation conditions, with tRNA binding eliciting kinase activation by weakening HisRS-CTD association with attendant disruption of the autoinhibitory KD-CTD interaction

An upstream ORF with non-AUG start codon is translated in vivo but dispensable for translational control of GCN4 mRNA

Genome-wide analysis of ribosome locations in mRNAs of Saccharomyces cerevisiae has revealed the translation of upstream open reading frames that initiate with near-cognate start codons in many transcripts. Two such non-translation initiation codon (AUG)-initiated upstream open reading frames (uORFs) (nAuORFs 1 and 2) occur in GCN4 mRNA upstream of the four AUG-initiated uORFs (uORFs 1–4) that regulate GCN4 translation. We verified that nAuORF2 is translated in vivo by demonstrating β-galactosidase production from lacZ coding sequences fused to nAuORF2, in a manner abolished by replacing its non-AUG initiation codon (AUA) start codon with the non-cognate triplet AAA, whereas translation of nAuORF1 was not detected. Importantly, replacing the near-cognate start codons of both nAuORFs with non-cognate triplets had little or no effect on the repression of GCN4 translation in non-starved cells, nor on its derepression in response to histidine limitation, nutritional shift-down or treatment with rapamycin, hydrogen peroxide or methyl methanesulfonate. Additionally, we found no evidence that initiation from the AUA codon of nAuORF2 is substantially elevated, or dependent on Gcn2, the sole eIF2α kinase of yeast, in histidine-deprived cells. Thus, although nAuORF2 is translated in vivo, it appears that this event is not stimulated by eIF2α phosphorylation nor significantly influences GCN4 translational induction under various starvation or stress conditions

Sect and House in Syria: History, Architecture, and Bayt Amongst the Druze in Jaramana

This paper explores the connections between the architecture and materiality of houses and the social idiom of bayt (house, family). The ethnographic exploration is located in the Druze village of Jaramana, on the outskirts of the Syrian capital Damascus. It traces the histories, genealogies, and politics of two families, bayt Abud-Haddad and bayt Ouward, through their houses. By exploring the two families and the architecture of their houses, this paper provides a detailed ethnographic account of historical change in modern Syria, internal diversity, and stratification within the intimate social fabric of the Druze neighbourhood at a time of war, and contributes a relational approach to the anthropological understanding of houses

GCN2-dependent phosphorylation of eukaryotic translation initiation factor-2α in Arabidopsis

The yeast regulatory protein kinase, general control non-derepressible-2 (GCN2) plays a key role in general amino acid control. GCN2 phosphorylates the α subunit of the trimeric eukaryotic translation initiation factor-2 (eIF2), bringing about a decrease in the general rate of protein synthesis but an increase in the synthesis of GCN4, a transcription factor that promotes the expression of genes encoding enzymes for amino acid biosynthesis. The present study concerned the phosphorylation of Arabidopsis eIF2α (AteIF2α) by the Arabidopsis homologue of GCN2, AtGCN2, and the role of AtGCN2 in regulating genes encoding enzymes of amino acid biosynthesis and responding to virus infection. A null mutant for AtGCN2 called GT8359 was obtained and western analysis confirmed that it lacked AtGCN2 protein. GT8359 was more sensitive than wild-type Arabidopsis to herbicides that affect amino acid biosynthesis. Phosphorylation of AteIF2α occurred in response to herbicide treatment but only in wild-type Arabidopsis, not GT8359, showing it to be AtGCN2-dependent. Expression analysis of genes encoding key enzymes for amino acid biosynthesis and nitrate assimilation revealed little effect of loss of AtGCN2 function in GT8359 except that expression of a nitrate reductase gene, NIA1, was decreased. Analysis of wild-type and GT8359 plants infected with Turnip yellow mosaic virus or Turnip crinkle virus showed that AteIF2α was not phosphorylated

Borrelia burgdorferi membranes are the primary targets of reactive oxygen species

Spirochetes living in an oxygen-rich environment or when challenged by host immune cells are exposed to reactive oxygen species (ROS). These species can harm/destroy cysteinyl residues, iron-sulphur clusters, DNA and polyunsaturated lipids, leading to inhibition of growth or cell death. Because Borrelia burgdorferi contains no intracellular iron, DNA is most likely not a major target for ROS via Fenton reaction. In support of this, growth of B. burgdorferi in the presence of 5 mM H2O2 had no effect on the DNA mutation rate (spontaneous coumermycin A1 resistance), and cells treated with 10 mM t-butyl hydroperoxide or 10 mM H2O2 show no increase in DNA damage. Unlike most bacteria, B. burgdorferi incorporates ROS-susceptible polyunsaturated fatty acids from the environment into their membranes. Analysis of lipoxidase-treated B. burgdorferi cells by Electron Microscopy showed significant irregularities indicative of membrane damage. Fatty acid analysis of cells treated with lipoxidase indicated that host-derived linoleic acid had been dramatically reduced (50-fold) in these cells, with a corresponding increase in the levels of malondialdehyde by-product (fourfold). These data suggest that B. burgdorferi membrane lipids are targets for attack by ROS encountered in the various stages of the infective cycle

Gut microbiota-derived propionate reduces cancer cell proliferation in the liver

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The battle over Syria's reconstruction

Reconstruction is becoming the new battleground in the Syrian conflict—its continuation by other means. It is instrumentalized by the regime as a way to reconsolidate its control over the country and by rival regional and international powers to shape the internal balance of power and establish spheres of influence in the country. The paper examines the Asad regime’s practices, including co-optation of militia leaders via reconstruction concessions and use of reconstruction to clear strategic areas of opposition-dominated urban settlements. The paper then surveys how the geopolitical struggle in Syria has produced an asymmetry as regards reconstruction: those powers that lost the geo-political contest on the ground seek to use geo-economic superiority to reverse the geo-political outcome. Then the impact of proxy wars and spheres of influence in the country on the security context for reconstruction is examined. Finally, the reconstruction initiatives of the various external parties are assessed, including Russia, Iran and Turkey as well as the spoiler role by which the US seeks to obstruct reconstruction that would spell victory in Syria for its Russian and Iranian rivals.PostprintPeer reviewe

The C-terminal domain of eukaryotic initiation factor 5 promotes start codon recognition by its dynamic interplay with eIF1 and eIF2 beta

Recognition of the proper start codon on mRNAs is essential for protein synthesis, which requires scanning and involves eukaryotic initiation factors (eIFs) eIF1, eIF1A, eIF2, and eIF5. The carboxyl terminal domain (CTD) of eIF5 stimulates 43S preinitiation complex (PIC) assembly; however, its precise role in scanning and start codon selection has remained unknown. Using nuclear magnetic resonance (NMR) spectroscopy, we identified the binding sites of eIF1 and eIF2β on eIF5-CTD and found that they partially overlapped. Mutating select eIF5 residues in the common interface specifically disrupts interaction with both factors. Genetic and biochemical evidence indicates that these eIF5-CTD mutations impair start codon recognition and impede eIF1 release from the PIC by abrogating eIF5-CTD binding to eIF2β. This study provides mechanistic insight into the role of eIF5-CTD's dynamic interplay with eIF1 and eIF2β in switching PICs from an open to a closed state at start codons.publishedVersio

Molecular Landscape of the Ribosome Pre-initiation Complex during mRNA Scanning: Structural Role for eIF3c and Its Control by eIF5

Citation: Obayashi, E., Luna, R. E., Nagata, T., Martin-Marcos, P., Hiraishi, H., Singh, C. R., . . . Asano, K. (2017). Molecular Landscape of the Ribosome Pre-initiation Complex during mRNA Scanning: Structural Role for eIF3c and Its Control by eIF5. Cell Reports, 18(11), 2651-2663. doi:10.1016/j.celrep.2017.02.052During eukaryotic translation initiation, eIF3 binds the solvent-accessible side of the 40S ribosome and recruits the gate-keeper protein eIF1 and eIF5 to the decoding center. This is largely mediated by the N-terminal domain (NTD) of eIF3c, which can be divided into three parts: 3c0, 3c1, and 3c2. The N-terminal part, 3c0, binds eIF5 strongly but only weakly to the ribosome-binding surface of eIF1, whereas 3c1 and 3c2 form a stoichiometric complex with eIF1. 3c1 contacts eIF1 through Arg-53 and Leu-96, while 3c2 faces 40S protein uS15/S13, to anchor eIF1 to the scanning pre-initiation complex (PIC). We propose that the 3c0:eIF1 interaction diminishes eIF1 binding to the 40S, whereas 3c0:eIF5 interaction stabilizes the scanning PIC by precluding this inhibitory interaction. Upon start codon recognition, interactions involving eIF5, and ultimately 3c0:eIF1 association, facilitate eIF1 release. Our results reveal intricate molecular interactions within the PIC, programmed for rapid scanning-arrest at the start codon