Total deletion of yeast LEU4: further evidence for a second alpha-isopropylmalate synthase and evidence for tight LEU4-MET4 linkage

Using a combination of restriction endonuclease digestion, nuclease BAL 31 treatment, and standard ligation procedures, a 4.4-kb DNA segment that carried the yeast LEU4 gene [encoding alpha-isopropylmalate synthase (IPMS) I] and adjoining sequences was excised from an appropriate plasmid and replaced with the yeast HIS3 gene. The new plasmid was digested to obtain a linear HIS3-carrying fragment flanked by remnants of the LEU4 region. Integrative transformation of a LEU4fbr LEU5+ his3- strain with this fragment resulted in the deletion of the LEU4 gene from the genome of some recipients, as demonstrated by transformant phenotype, genetic analysis and the absence of RNA capable of hybridizing to a LEU4 probe. The leu4 deletion strains remained Leu+. The extract of one such strain contained about 18% of the IPMS activity of wild-type cells. It is concluded that the residual activity is that of a second IPMS (IPMS II) that depends on an intact LEU5 locus. IPMS II was inhibited by leucine, but its sensitivity was about an order of magnitude lower than that of IPMS I. Deletion of the LEU4 region by the method utilized here resulted in an amino acid auxotrophy that could be satisfied by methionine, homocysteine, or cysteine. Complementation tests and genetic analysis demonstrated that the affected gene was MET4. Linkage to MET4 would place the LEU4 gene on the left arm of chromosome XIV.     

Gene-specific translational control of the yeast GCN4 gene by phosphorylation of eukaryotic initiation factor 2

Phosphorylation of the α subunit of eukaryotic initiation factor 2 (elF-2α) is one of the best-characterized mechanisms for down-regulating total protein synthesis in mammalian cells in response to various stress conditions. Recent work indicates that regulation of the GCN4 gene of Saccharomyces cerevisiae by amino acid availability represents a gene-specific case of translational control by phosphorylation of elF-2α, Four short open reading frames in the leader of GCN4 mRNA (uORFs) restrict the flow of scanning ribosomes from the cap site to the GCN4 initiation codon. When amino acids are abundant, ribosomes translate the first uORF and reinitiate at one of the remaining uORFs in the leader, after which they dissociate from the mRNA. Under conditions of amino acid starvation, many ribosomes which have translated uORFI fail to reinitiate at uORFs 2-4 and utilize the GCN4 start codon instead. Failure to reinitiate at uORFs 2-4 in starved cells results from a reduction in the GTP-bound form of elF-2 that delivers charged initiator tRNA_i^Met to the ribosome. When the levels of elF-2·GTP·Met-tRNA_i^Met ternary complexes are low, many ribosomes will not rebind this critical initiation factor following translation of uORF1 until after scanning past uORF4, but before reaching GCN4. Phosphorylation of elF-2 by the protein kinase GCN2 decreases the concentration of elF-2·GTP·Met-tRNA_i^Met complexes by inhibiting the guanine nucleotide exchange factor for elF-2, which is the same mechanism utilized in mammalian cells to inhibit total protein synthesis by phosphorylation of elF-2.     

Evidence for a role of topoisomerase II in the Ca2+-dependent basal level expression of the rat prolactin gene

The PRL gene is expressed at a high basal level in rat pituitary tumor GH3 cells, and this basal level enhancement of PRL gene expression is maintained through a Ca2+-calmodulin-dependent mechanism. We have now examined whether the enzyme, DNA topoisomerase II, which has been shown to be phosphorylated by a Ca2+-calmodulin-dependent protein kinase, plays a role in the Ca2+-calmodulin-dependent basal level enhancement of PRL gene expression. The topoisomerase II inhibitor, novobiocin, at concentrations in the range of 35-140 microM, effectively blocked the ability of Ca2+ to increase PRL mRNA levels. Examination of the effects of novobiocin on the levels of protein synthesis, glucose-regulated protein (GRP) 78 mRNA, histone 3 mRNA, and 18S ribosomal RNA indicated that the drug selectivity inhibited PRL gene expression. Two other topoisomerase II inhibitors, m-AMSA and VM26, also diminished the Ca2+-induced levels of PRL mRNA at concentrations (100-400 nM) that did not lower total mRNA levels. We then examined whether topoisomerase II interacted nonrandomly with DNA from the 5' transcribed and 5'-flanking region of the rat PRL gene by in vitro mapping of topoisomerase II DNA cleavage sites. In initial assays with a 10.5 kilobase (kb) PRL genomic DNA fragment containing 3.5 kb of 5'-transcribed DNA and 7 kb of 5'-flanking DNA, we detected 4 major cleavage sites in the following regions: site 1, +1500 to +1600; site 2, +1 to -100; site 3, -1200 to -1300; and site 4, -2900 to -3000.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multicopy tRNA genes functionally suppress mutations in yeast eIF-2 alpha kinase GCN2: evidence for separate pathways coupling GCN4 expression to unchanged tRNA.

GCN2 is a protein kinase that stimulates translation of GCN4 mRNA in amino acid-starved cells by phosphorylating the alpha subunit of translation initiation factor 2 (eIL-2). We isolated multicopy plasmids that overcome the defective derepression of GCN4 and its target genes caused by the leaky mutation gcn2-507. One class of plasmids contained tRNA(His) genes and conferred efficient suppression only when cells were starved for histidine; these plasmids suppressed a gcn2 deletion much less efficiently than they suppressed gcn2-507. This finding indicates that the reduction in GCN4 expression caused by gcn2-507 can be overcome by elevating tRNA(His) expression under conditions in which the excess tRNA cannot be fully aminoacylated. The second class of suppressor plasmids all carried the same gene encoding a mutant form of tRNA(Val) (AAC) with an A-to-G transition at the 3' encoded nucleotide, a mutation shown previously to reduce aminoacylation of tRNA(Val) in vitro. In contrast to the wild-type tRNA(His) genes, the mutant tRNA(Val) gene efficiently suppressed a gcn2 deletion, and this suppression was independent of the phosphorylation site on eIF-2 alpha (Ser-51). Overexpression of the mutant tRNA(Val) did, however, stimulate GCN4 expression at the translational level. We propose that the multicopy mutant tRNA(Val) construct leads to an accumulation of uncharged tRNA(Val) that derepresses GCN4 translation through a pathway that does not involve GCN2 or eIF-2 alpha phosphorylation. This GCN2-independent pathway was also stimulated to a lesser extent by the multicopy tRNA(His) constructs in histidine-deprived cells. Because the mutant tRNA(Val) exacerbated the slow-growth phenotype associated with eIF-2 alpha hyperphosphorylation by an activated GCN2c kinase, we suggest that the GCN2-independent derepression mechanism involves down-regulation of eIF-2 activity.