Previously we found that a mutation in either pgi or pfkA, encoding phosphoglucose isomerase or phosphofructokinase A, respectively, facilitates degradation of the ptsG mRNA in an RNase E-dependent manner in Escherichia coli (1). In this study, we examined the effects of a series of glycolytic genes on the degradation of ptsG mRNA and how the mutations destabilize the ptsG mRNA. The conditional lethal mutation ts8 in fda, encoding fructose-1,6-P 2 aldolase just downstream of pfkA in the glycolytic pathway, caused the destabilization of ptsG mRNA at the nonpermissive temperature. Mutations in any other gene did not destabilize the ptsG mRNA; rather, they reduced the ptsG transcription mainly by affecting the cAMP level. The rapid degradation of ptsG mRNA in mutant strains was completely dependent upon the presence of glucose or any one of its compounds, which enter the Embden-Meyerhof glycolytic pathway before the block points. A significant increase in the intracellular glucose-6-P level was observed in the presence of glucose in the pgi strain. An overexpression of glucose-6-phosphate dehydrogenase eliminated both the accumulation and the degradation of ptsG mRNA in the pgi strain. In addition, accumulation of fructose-6-P led to the rapid degradation of ptsG mRNA in a pgi pfkA mutant strain lacking glucose-6-P. We conclude that the RNase E-dependent destabilization of ptsG mRNA occurs in response to accumulation of glucose-6-P or fructose-6-P.In bacteria, a number of sugars represented by glucose are transported into the cells coupled with their phosphorylation by the phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS) 1 (2-4), whereas the translocation of some other sugars such as lactose is catalyzed by non-PTS transport systems. In either case, the incorporated sugars are metabolized primarily by the Embden-Meyerhof glycolytic pathway and by the pentose phosphate pathway to produce numerous intermediary metabolites as well as energy in cells (5). The PTS in Escherichia coli consists of two common cytoplasmic proteins, enzyme I and HPr (histidine-containing protein of the PTS), as well as an array of sugar-specific enzyme II complexes (EIIs). The glucose-specific EII (glucose transporter) consists of cytoplasmic protein IIA Glc and membrane receptor IICB Glc encoded by crr and ptsG, respectively. The phosphoryl group from phosphoenolpyruvate is transferred sequentially to enzyme I, HPr, the EIIs, and finally glucose as it is translocated across the membrane.In addition to sugar transport and phosphorylation, the PTS plays important regulatory roles in a variety of cellular activities. This is particularly evident for the glucose-specific PTS. For example, IIA Glc regulates both the transport of non-PTS sugars and the activity of adenylate cyclase depending on its phosphorylation state (2-4). The former process, called inducer exclusion, is fully responsible for the glucose-lactose diauxie that is a prototype of catabolite repression (6, 7). A striking recent discovery regarding the regulatory...

External glucose stimulates transcription of several genes including ptsG encoding IICB(Glc), a membrane component of the phosphotransferase system (PTS), by relieving the negative regulation of a global repressor Mlc in Escherichia coli. We investigate here how glucose modulates Mlc action. The Mlc-mediated repression is eliminated by a ptsI mutation, while Mlc is constitutively active in a ptsG mutant. We show that IICB(Glc)-FLAG interacts physically with Mlc in crude extracts prepared from cells in which IICB(Glc) is supposed to exist as the non-phosphorylated form. The IICB(Glc)-Mlc interaction is no longer observed when IICB(Glc) is phosphorylated. Exogenously added purified Mlc binds to purified IICB(Glc)-FLAG. We also demonstrate that Mlc is associated with membrane when IICB(Glc) is dephosphorylated while it is in the cytoplasm when IICB(Glc) is phosphorylated or absent. We conclude that IICB(Glc) regulates the cellular localization of Mlc, depending on its phosphorylation state, which is determined by the availability of external glucose. Thus, glucose induces the transcription of Mlc-regulated promoters by sequestering Mlc to the membrane through dephosphorylation of IICB(Glc).

The large protein kinases, ataxia-telangiectasia mutated (ATM) and ATM-Rad3-related (ATR), coordinate the cellular response to DNA damage. In budding yeast, ATR homologue Mec1 plays a central role in DNA damage signaling. Mec1 interacts physically with Ddc2 and functions in the form of the Mec1-Ddc2 complex. To identify proteins interacting with the Mec1-Ddc2 complex, we performed a modified two-hybrid screen and isolated RFA1 and RFA2, genes that encode subunits of replication protein A (RPA). Using the two-hybrid system, we found that the extreme C-terminal region of Mec1 is critical for RPA binding. The C-terminal substitution mutation does not affect the Mec1-Ddc2 complex formation, but it does impair the interaction of Mec1 and Ddc2 with RPA as well as their association with DNA lesions. The C-terminal mutation also decreases Mec1 kinase activity. However, the Mec1 kinase-defect by itself does not perturb Mec1 association with sites of DNA damage. We also found that Mec1 and Ddc2 associate with sites of DNA damage in an interdependent manner. Our findings support the model in which Mec1 and Ddc2 localize to sites of DNA damage by interacting with RPA in the form of the Mec1-Ddc2 complex. INTRODUCTIONThe maintenance of genome stability is critical to cellular survival and proliferation in all organisms. Cells have evolved surveillance mechanisms that monitor genomic lesions and activate various DNA damage responses, including cell cycle arrest and transcriptional induction of DNA repair genes (Zhou and Elledge, 2000). This surveillance mechanism is called DNA damage checkpoint in eukaryotes. The checkpoint signals are initiated through two large protein kinases, ataxia-telangiectasia mutated (ATM) and ATM-Rad3-related (ATR) (Zhou and Elledge, 2000;Abraham, 2001). ATM and ATR are highly conserved among eukaryotes. ATR is closely related to Mec1 in the budding yeast Saccharomyces cerevisiae and Rad3 in the fission yeast Schizosaccharomyces pombe. ATM homologues are termed Tel1 in both budding and fission yeasts.In the budding yeast S. cerevisiae, Mec1 plays a central role in DNA damage checkpoint control, whereas Tel1 plays a minor role (Morrow et al., 1995;Sanchez et al., 1996;Usui et al., 2001;Nakada et al., 2003bNakada et al., , 2004. Mec1 physically interacts with Ddc2 (also called Lcd1 and Pie1), a protein that exhibits homology to the ATR-interacting protein ATRIP and Rad3-interacting protein Rad26 (Edwards et al., 1999;Paciotti et al., 2000;Rouse and Jackson, 2000;Cortez et al., 2001;Wakayama et al., 2001). Mec1 and Ddc2 function in the form of the Mec1-Ddc2 complex, and both localize to sites of DNA damage Melo et al., 2001;Rouse and Jackson, 2002). Mec1 controls two downstream protein kinases Chk1 and Rad53, which are related to mammalian Chk1 and Chk2, respectively (Zhou and Elledge, 2000). Rad53 plays a central role in DNA damage checkpoints throughout the cell cycle (Longhese et al., 1998), whereas Chk1 acts in part at G 2 /M (Sanchez et al., 1999). Rad53 becomes phosphorylated and activated after ...

Background: The pts operon of Escherichia coli consists of three genes ptsH, ptsI and crr, each encoding for central components of the phosphoenolpyruvate: carbohydrate phosphotransferase system, HPr, enzyme I and IIA

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