Previous studies in Saccharomyces cerevisiae have demonstrated that cryptic promoters within coding regions activate transcription in particular mutants. We have performed a comprehensive analysis of cryptic transcription in order to identify factors that normally repress cryptic promoters, to determine the amount of cryptic transcription genome-wide, and to study the potential for expression of genetic information by cryptic transcription. Our results show that a large number of factors that control chromatin structure and transcription are required to repress cryptic transcription from at least 1,000 locations across the S. cerevisiae genome. Two results suggest that some cryptic transcripts are translated. First, as expected, many cryptic transcripts contain an ATG and an open reading frame of at least 100 codons. Second, several cryptic transcripts are translated into proteins. Furthermore, a subset of cryptic transcripts tested is transiently induced in wild-type cells following a nutritional shift, suggesting a possible physiological role in response to a change in growth conditions. Taken together, our results demonstrate that, during normal growth, the global integrity of gene expression is maintained by a wide range of factors and suggest that, under altered genetic or physiological conditions, the expression of alternative genetic information may occur.

We investigated the timing of the recruitment of Spn1 and its partner, Spt6, to the CYC1 gene. Like TATA binding protein and RNA polymerase II (RNAPII), Spn1 is constitutively recruited to the CYC1 promoter, although levels of transcription from this gene, which is regulated postrecruitment of RNAPII, are low. In contrast, Spt6 appears only after growth in conditions in which the gene is highly transcribed. Spn1 recruitment is via interaction with RNAPII, since an spn1 mutant defective for interaction with RNAPII is not targeted to the promoter, and Spn1 is necessary for Spt6 recruitment. Through a targeted genetic screen, strong and specific antagonizing interactions between SPN1 and genes encoding Swi/Snf subunits were identified. Like Spt6, Swi/Snf appears at CYC1 only after activation of the gene. However, Spt6 significantly precedes Swi/Snf occupancy at the promoter. In the absence of Spn1 recruitment, Swi/Snf is constitutively found at the promoter. These observations support a model whereby Spn1 negatively regulates RNAPII transcriptional activity by inhibiting recruitment of Swi/Snf to the CYC1 promoter, and this inhibition is abrogated by the Spn1-Spt6 interaction. These findings link Spn1 functions to the transition from an inactive to an actively transcribing RNAPII complex at a postrecruitment-regulated promoter.For a large number of well-characterized genes, the ratelimiting step in the transcription process is the formation of the preinitiation complex at the promoter. At these genes, the recruitment of TATA binding protein (TBP) and RNA polymerase II (RNAPII) to the promoter correlates strongly with transcriptional output (35,41,67). Indeed, delivery of TBP and RNAPII appears to be sufficient for gene activation in many contexts (for reviews, see references 63 and 64). However, there are a growing number of genes that have been found to be regulated at a step after the recruitment of RNAPII. Such genes include the yeast CYC1 gene, the Drosophila heat shock genes, and mammalian human immunodeficiency virus type 1 and the c-Myc proto-oncogene (3,39,41,51,77). Indeed, whole-genome studies suggest that a significant portion of the human genome may be regulated postrecruitment of RNAPII (26,35). As such, these mechanisms have the potential to impact the expression of thousands of human genes. Our understanding of these mechanisms and the rate-limiting steps involved is incomplete, but these observations suggest that functions critical for a high level of transcription are either inhibited or absent under noninducing conditions. To determine the nature of these functions, further characterization of genes regulated after the assembly of the general transcription machinery is imperative.The yeast CYC1 gene encodes iso-1-cytochrome c, a protein involved in the electron transport chain in the mitochondria (75). In the presence of a fermentable carbon source (such as dextrose), CYC1 gene expression is inhibited and transcriptional levels are extremely low (24,25). When cells are grown on a nonfermentable c...

The spatial activation of phosphoinositide 3-kinase (PI3-kinase) signaling at the axon growth cone generates phosphatidylinositol 3,4,5 trisphosphate (PtdIns(3,4,5)P3), which localizes and facilitates Akt activation and stimulates GSK-3beta inactivation, promoting microtubule polymerization and axon elongation. However, the molecular mechanisms that govern the spatial down-regulation of PtdIns(3,4,5)P3 signaling at the growth cone remain undetermined. The inositol polyphosphate 5-phosphatases (5-phosphatase) hydrolyze the 5-position phosphate from phosphatidylinositol 4,5 bisphosphate (PtdIns(4,5)P2) and/or PtdIns(3,4,5)P3. We demonstrate here that PIPP, an uncharacterized 5-phosphatase, hydrolyzes PtdIns(3,4,5)P3 forming PtdIns(3,4)P2, decreasing Ser473-Akt phosphorylation. PIPP is expressed in PC12 cells, localizing to the plasma membrane of undifferentiated cells and the neurite shaft and growth cone of NGF-differentiated neurites. Overexpression of wild-type, but not catalytically inactive PIPP, in PC12 cells inhibited neurite elongation. Targeted depletion of PIPP using RNA interference (RNAi) resulted in enhanced neurite differentiation, associated with neurite hyperelongation. Inhibition of PI3-kinase activity prevented neurite hyperelongation in PIPP-deficient cells. PIPP targeted-depletion resulted in increased phospho-Ser473-Akt and phospho-Ser9-GSK-3beta, specifically at the neurite growth cone, and accumulation of PtdIns(3,4,5)P3 at this site, associated with enhanced microtubule polymerization in the neurite shaft. PIPP therefore inhibits PI3-kinase-dependent neurite elongation in PC12 cells, via regulation of the spatial distribution of phospho-Ser473-Akt and phospho-Ser9-GSK-3beta signaling.

The malaria parasite Plasmodium falciparum relies on efficient protein translation. An essential component of translation is the tryptophanyl-tRNA synthetase (TrpRS) that charges tRNAtrp. Here we characterise two isoforms of TrpRS in Plasmodium; one eukaryotic type localises to the cytosol and a bacterial type localises to the remnant plastid (apicoplast). We show that the apicoplast TrpRS aminoacylates bacterial tRNAtrp while the cytosolic TrpRS charges eukaryotic tRNAtrp. An inhibitor of bacterial TrpRSs, indolmycin, specifically inhibits aminoacylation by the apicoplast TrpRS in vitro, and inhibits ex vivo Plasmodium parasite growth, killing parasites with a delayed death effect characteristic of apicoplast inhibitors. Indolmycin treatment ablates apicoplast inheritance and is rescuable by addition of the apicoplast metabolite isopentenyl pyrophosphate (IPP). These data establish that inhibition of an apicoplast housekeeping enzyme leads to loss of the apicoplast and this is sufficient for delayed death. Apicoplast TrpRS is essential for protein translation and is a promising, specific antimalarial target.

No abstract