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The human p68 kinase is an interferon‐regulated enzyme that inhibits protein synthesis when activated by double‐stranded RNA. We show here that when expressed in Saccharomyces cerevisiae, the p68 kinase produced a growth suppressing phenotype resulting from an inhibition of polypeptide chain initiation consistent with functional protein kinase activity. This slow growth phenotype was reverted in yeast by two different mechanisms: expression of the p68 kinase N‐terminus, shown to bind double‐stranded RNA in vitro and expression of a mutant form of the alpha‐subunit of yeast initiation factor 2, altered at a single phosphorylatable site. These results provide the first direct in vivo evidence that the p68 kinase interacts with the alpha‐subunit of eukaryotic initiation factor 2. Sequence similarity with a yeast translational regulator, GCN2, further suggests that this enzyme may be a functional homolog in higher eukaryotes, where its normal function is to regulate protein synthesis through initiation factor 2 phosphorylation.

Phosphorylation of the a subunit of eukaryotic initiation factor 2 (eIF-2a) in Saccharomyces cerevisiae by the GCN2 protein kinase stimulates the translation of GCN4 mRNA. The protein kinases heme-regulated inhibitor of translation (HRI) and double-stranded RNA-dependent eIF-2a protein kinase (dsRNA-PK) inhibit initiation of translation in mammalian cells by phosphorylating Ser-51 of eIF-2a. We show that HRI and dsRNA-PK phosphorylate yeast eIF-2a in vitro and in vivo and functionally substitute for GCN2 protein to stimulate GCN4 translation in yeast. In addition, high-level expression of either mammalian kinase in yeast decreases the growth rate, a finding analogous to the inhibition of total protein synthesis by these kinases in mammalian cells. Phosphorylation of eIF-2a inhibits initiation in mammalian cells by sequestering eIF-2B, the factor required for exchange of GTP for GDP on eIF-2. Mutations in the GCN3 gene, encoding a subunit of the yeast eIF-2B complex, eliminate the effects of HRI and dsRNA-PK on global and GCN4-specific translation in yeast. These results provide further in vivo evidence that phosphorylation of eIF-2a inhibits translation by impairing eIF-2B function and identify GCN3 as a regulatory subunit of eIF-2B. These results also suggest that GCN4 translational control will be a good model system to study how mammalian eIF-2a kinases are modulated by environmental signals and viral regulatory factors.Mammalian cells use translational control mechanisms for a rapid response to various types of stress. The protein kinases heme-regulated inhibitor of translation (HRI) and doublestranded RNA-dependent a subunit of eukaryotic initiation factor 2 (eIF-2a) kinase (dsRNA-PK; also referred to as p68 kinase, DAI, or dsl) are activated by heme-deprivation and virus infection, respectively, to inhibit total protein synthesis by phosphorylating Ser-51 of eIF-2a (for review, see refs. 1 and 2). Composed of three subunits, eIF-2 forms a ternary complex with GTP and charged initiator tRNAMet (MettRNAMet) and functions to deliver the Met-tRNAMet to the ribosome during translation initiation. The GTP ofthe ternary complex is hydrolyzed to GDP during translation initiation, and eIF-2 is released from the ribosome as an eIF-2-GDP complex. A second factor, eIF-2B, is required to catalyze the exchange of GTP for GDP in the eIF-2-GDP complex. Phosphorylation of eIF-2a on Ser-51 inhibits translation initiation by sequestering eIF-2B (2, 3). This inhibition of eIF-2B activity diminishes the recycling of eIF-2-GDP to eIF-2-GTP, decreasing ternary complex formation and reducing the rate of initiation of translation.In the yeast Saccharomyces cerevisiae, phosphorylation of eIF-2a by the protein kinase GCN2 mediates translational control of the GCN4 gene. GCN4 is a transcriptional activator of amino acid biosynthetic genes that must be expressed at high levels to overcome the effects of amino acid starvation. GCN4 expression is regulated at the translational level by four short open reading frames (ORFs) in th...

The properties of a new bis(platinum) complex containing two monodentate coordination spheres, [(trans-PtCl(NH3)2)2H2N(CH2)4NH2]Cl2 (1,1/t,t), are reported. Comparison is made with respect to chemical reactivity, in vitro biological activity in murine and tumor cells, DNA conformational changes, cross-linking efficiency, and sequence specificity between this complex and the previously reported complex containing two bidentate platinum atoms, [(Pt(mal)(NH3))2H2N(CH2)4NH2] (2,2/c,c), as well as with their respective monomeric analogues, [PtCl(dien)]Cl and cis-[PtCl2(NH3)2](cis-DDP). While both bis(platinum) complexes are active against cis-DDP-resistant cells, the monodentate bis(platinum) complex (1,1/t,t) has a lower resistance factor than the complex with bidentate coordination spheres (2,2/c,c). More importantly, this property is repeated in a human ovarian carcinoma cell line. DNA-binding studies show that DNA interstrand cross-linking is more efficient for the 1,1/t,t complex. DNA sequencing studies employing the exonuclease activity of T4-polymerase demonstrate that there are a variety of binding sites; some are common to all complexes and some common to both bis(platinum) complexes, while the monodentate 1,1/t,t species also reacts at unique sites, not attacked by any of the other complexes studied. The circular dichroism of CT DNA modified by the 1,1/t,t complex is also unique and is not seen for any of the other agents.

Genetic reversion of HIS4 initiator codon mutations in yeast has identified three unlinked genes, suil, sui2, and SUI3 (suppressors of initiator codon mutations), which when mutated confer the ability to initiate translation at HIS4 despite the absence of an AUG start codon. We have previously demonstrated that the SUI3 gene encodes the j3 subunit of the eukaryotic initiation factor 2 (eIF-2) and that mutations at a Zn(II) finger motif of SUI3 alter the start site selection process in yeast. In this report, molecular and biochemical characterizations show that the sui2 suppressor gene encodes the a subunit of eIF-2. The amino acid sequence of sui2 is 58% homologous to that encoded by the cDNA of the human eIF-2a. Mutations in the sui2 suppressor alleles occur in the amino-terminal portion of the protein and change amino acids that are identical at the same relative position in the yeast and human proteins. Protein sequence analysis shows that a sui2 mutant yeast strain allows initiation at a UUG codon in the absence of an AUG codon at HIS4. These data further suggest that eIF-2 is an important component of the preinitiation complex that mediates ribosomal recognition of a start codon during the scanning process.The eukaryotic translation initiation factor 2 (eIF-2) is composed of three nonidentical subunits: a, P3, and 'y(1). One role of eIF-2 established by biochemical studies (2) is that it functions during the early steps of translation initiation by forming a ternary complex with GTP and initiator tRNA. This complex then binds the small 40S ribosomal subunit, which in turn binds the 5' end of eukaryotic mRNA. According to the scanning model (3, 4), this preinitiation complex then scans the leader region until the first AUG codon is reached whereupon translation begins.Recent genetic studies in our laboratory have provided biological evidence that eIF-2 may also function in ribosomal selection of the initiator codon during t1e scanning process (5). By reverting his4-, his4-lacZ Saccharomyces cerevisiae initiator codon mutants (His-, white), three unlinked genes, suil, sui2, and SUI3, were identified that when mutated act in trans to restore both his4 and his4-lacZ expression [His', blue revertant colonies on synthetic dextrose minus histidine and 5-bromo-4-chloro-3-indolyl-,8-D-galactoside (X-Gal) In this report, we present the characterization of the sui2 suppressor gene. Our study shows that sui2 encodes the a subunit of eIF-2 and mutations in a also confer an alteration in start site selection by allowing initiation at a UUG codon in the mutant his4 message. As observed for the yeast and human P3 subunit of eIF-2, the yeast a subunit shows considerable identity to the amino acid sequence derived from a cDNA encoding the human a subunit of eIF-2 (7) and mutations in sui2 suppressor genes that restore his4 expression change amino acids that are identical at the same position in the human eIF-2a sequence. In light of similarities between the yeast and mammalian initiation processes (3,4,8,9), these ...

The mechanism by which the scanning ribosome recognizes the first AUG codon nearest the 5' end of eukaryotic messenger RNA has not been established. To investigate this an anticodon change (3'-UCC-5') was introduced into one of the four methionine initiator (tRNAi(met) genes of Saccharomyces cerevisiae. The ability of the mutant transfer RNA to restore growth properties to his4 initiator codon mutant yeast strains in the absence of histidine was then assayed. Only the complementary codon, AGG, at the his4 initiator region supported His+ growth. The mutant transfer RNA also directed the ribosome to initiate at an AGG placed in the upstream region of the his4 message. Initiation at this upstream AGG precluded initiation at a downstream AGG in accordance with the "scanning" model. Therefore, an anticodon: codon interaction between tRNAi(met) as part of the scanning ribosome and the first AUG must function in directing the ribosome to the eukaryotic initiator region.

We previously identified a rearrangement of mixed-lineage leukemia (MLL) gene (also known as ALL-1, HRX, and HTRX1), consisting of an in-frame partial tandem duplication (PTD) of exons 5 through 11 in the absence of a partner gene, occurring in approximately 4%-7% of patients with acute myeloid leukemia (AML) and normal cytogenetics, and associated with a poor prognosis. The mechanism by which the MLL PTD contributes to aberrant hematopoiesis and/or leukemia is unknown. To examine this, we generated a mouse knockin model in which exons 5 through 11 of the murine Mll gene were targeted to intron 4 of the endogenous Mll locus. Mll PTD/WT mice exhibit an alteration in the boundaries of normal homeobox (Hox) gene expression during embryogenesis, resulting in axial skeletal defects and increased numbers of hematopoietic progenitor cells. Mll PTD/WT mice overexpress Hoxa7, Hoxa9, and Hoxa10 in spleen, BM, and blood. An increase in histone H3/H4 acetylation and histone H3 lysine 4 (Lys4) methylation within the Hoxa7 and Hoxa9 promoters provides an epigenetic mechanism by which this overexpression occurs in vivo and an etiologic role for MLL PTD gain of function in the genesis of AML.

Previous studies have demonstrated that the a subunit of eukaryotic initiation factor 2 (eIF-2a), encoded by the SUI2 gene in the yeast Saccharomyces cerevisiae, is phosphorylated at Ser-51 by the GCN2 kinase in response to general amino acid control. Here we describe that yeast eIF-2a is a constitutively phosphorylated protein species that is multiply phosphorylated by a GCN2-independent mechanism. 32p; labeling and isoelectric focusing analysis of a SUI2+ Agcn2 strain identifies eIF-2ot as radiolabeled and a single isoelectric protein species. Treatment of SUI2+ Agcn2 strain extracts with phosphatase results in the identification of three additional isoelectric forms of eIF-2a that correspond to the stepwise removal of three phosphates from the protein. These data strongly support the conclusion that casein kinase II directly phosphorylates eIF-2oa at one or all of these Ser amino acids in vivo. Although substitution of SUI2 genes mutated at these sites for the wild-type gene have no obvious effect on cell growth, one test that we have used appears to demonstrate that the inability to phosphorylate these sites has a physiological consequence on eIF-2 function in S. cerevisiae. Haploid strains constructed to contain Ser-to-Ala mutations at the consensus casein kinase II sequences in SUI2 in combination with a mutated allele of either the GCN2, GCN3, or GCD7 gene have synthetic growth defects. These genetic data appear to indicate that the modifications that we describe at the carboxyl end of the eIF-2a protein are required for optimal eIF-2 function in S. cerevisiae.Eukaryotic translation initiation factor 2 (eIF-2) has been extensively characterized at the biochemical and genetic levels. Biochemical studies have established eIF-2 to be composed of three nonidentical subunits, cx, 1, and y, that function during the early steps of translation initiation by forming a ternary complex with GTP and the initiator tRNA (reviewed in references 29 and 39). This complex then binds the 40S ribosomal subunit, which in turn binds the 5' end of mRNA. According to the scanning model, this preinitiation complex scans the leader region until the first AUG codon is reached, whereupon translation begins (reviewed in references 33 and 34). Genetic studies from our laboratory have implicated eIF-2 to also play a role in ribosomal recognition of an AUG start codon. By reverting his4-initiator codon mutants, three unlinked genes, suil, sui2, and SUI3, were identified that when mutated act in trans to restore his4 expression (12). Characterization of the SUI2 and SUI3 genes showed that they encoded the a and a subunits of eIF-2, which are 42 and 58% identical in amino acid sequence to the human eIF-2oa and -13 proteins, respectively (17,25). Further analysis demonstrated that suppressor mutations in these genes conferred the ability to the ribosome to initiate translation at a UUG codon in the early his4 coding region by allowing a mismatched base pair interaction between the UUG codon and the initiator tRNA (17,25,63). The mutations ...