The RNA polymerase II holoenzyme consists of RNA polymerase II, a subset of general transcription factors, and regulatory proteins known as SRB proteins. The genes encoding SRB proteins were isolated as suppressors of mutations in the RNA polymerase II carboxy-terminal domain (CTD). The CTD and SRB proteins have been implicated in the response to transcriptional regulators. We report here the isolation of two new SRB genes, SRB10 and SRB11, which encode kinase- and cyclin-like proteins, respectively. Genetic and biochemical evidence indicates that the SRB10 and SRB11 proteins form a kinase-cyclin pair in the holoenzyme. The SRB10/11 kinase is essential for a normal transcriptional response to galactose induction in vivo. Holoenzymes lacking SRB10/11 kinase function are strikingly deficient in CTD phosphorylation. Although defects in the kinase substantially affect transcription in vivo, purified holoenzymes lacking SRB10/11 kinase function do not show defects in defined in vitro transcription systems, suggesting that the factors necessary to elicit the regulatory role of the SRB10/11 kinase are missing in these systems. These results indicate that the SRB10/11 kinase is involved in CTD phosphorylation and suggest that this modification has a role in the response to transcriptional regulators in vivo.
Deacidification of grape musts is crucial for the production of well-balanced wines, especially in colder regions of the world. The major acids in wine are tartaric and malic acid. Saccharomyces cerevisiae cannot degrade malic acid efficiently due to the lack of a malate transporter and the low substrate affinity of its malic enzyme. We have introduced efficient pathways for malate degradation in S. cerevisiae by cloning and expressing the Schizosaccharomyces pombe malate permease (mae1) gene with either the S. pombe malic enzyme (mae2) or Lactococcus lactis malolactic (mleS) gene in this yeast. Under aerobic conditions, the recombinant strain expressing the mae1 and mae2 genes efficiently degraded 8 g/L of malate in a glycerol-ethanol medium within 7 days. The recombinant malolactic strain of S. cerevisiae (mae1 and mleS genes) fermented 4.5 g/L of malate in a synthetic grape must within 4 days.
Sequence analysis of a 4.6-kb HindIII fragment containing the malic enzyme gene (mae2) of Schizosaccharomyces pombe, revealed the presence of an open reading frame of 1695 nucleotides, coding for a 565 amino acid polypeptide. The mae2 gene is expressed constitutively and encodes a single mRNA transcript of 2.0 kb. The mae2 gene was mapped on chromosome III by chromoblotting. The coding region and inferred amino acid sequence showed significant homology with 12 malic enzyme genes and proteins from widely different origins. Eight highly homologous regions were found in these malic enzymes, suggesting that they contain functionally conserved amino acid sequences that are indispensable for activity of malic enzymes. Two of these regions have previously been reported to be NAD- and NADP-binding sites.
The NAD-dependent malic enzyme from Schizosaccharomyces pombe catalyzes the oxidative decarboxylation of L-malate to pyruvate and CO 2 . Transcription of the S. pombe malic enzyme gene, mae2, was studied to elucidate the regulatory mechanisms involved in the expression of the gene. No evidence for substrate-induced expression of mae2 was observed in the presence of 0.2% L-malate. However, transcription of mae2 was induced when cells were grown in high concentrations of glucose or under anaerobic conditions. The increased levels of malic enzyme may provide additional pyruvate or assist in maintaining the redox potential under fermentative conditions. Deletion and mutation analyses of the 5-flanking region of the mae2 gene revealed the presence of three novel negative cis-acting elements, URS1, URS2, and URS3, that seem to function cooperatively to repress transcription of the mae2 gene. URS1 and URS2 are also present in the promoter region of the S. pombe malate transporter gene, suggesting co-regulation of their expression. Furthermore, two positive cisacting elements in the mae2 promoter, UAS1 and UAS2, show homology with the DNA recognition sites of the cAMP-dependent transcription factors ADR1, AP-2, and ATF (activating transcription factor)/CREB (cAMP response element binding).The fission yeast Schizosaccharomyces pombe efficiently degrades L-malate to CO 2 under aerobic conditions and to ethanol and CO 2 under anaerobic conditions (1). Cells of S. pombe are not able to utilize malate as the sole energy source or incorporate the malate into biomass (2) and therefore require glucose or other carbon sources for the energy-dependent transport and efficient degradation of malic acid (3). Three enzymes are involved in malate degradation in S. pombe, namely the malate transporter, malic enzyme, and malate dehydrogenase (4). The transporter, encoded by the mae1 gene (5), uses an H ϩ -symport system for the active transport of L-malate, and the NAD-dependent malic enzyme (EC 1.1.1.38) catalyzes the oxidative decarboxylation of L-malate to pyruvate and CO 2 . The mitochondrial malate dehydrogenase oxidizes L-malate to oxaloacetate in the tricarboxylic acid cycle and is responsible for 10% of the degradation of malate under aerobic conditions. Molecular analysis of the S. pombe malic mae2 gene showed a high degree of homology with malic enzymes from various organisms (6). Eight highly conserved regions were identified in malic enzymes, including the binding sites for L-malate and the dinucleotide co-factors NAD(P) ϩ (7,8). Although the secondary structure of malic enzymes is highly conserved, the coenzyme specificity (NAD ϩ or NADP ϩ ) and cellular localization (cytosolic or mitochondrial) are strongly linked to their regulation and metabolic function (9). Cytosolic NADP-dependent malic enzymes play an important role in lipid metabolism in higher eukaryotes (10), whereas NAD-dependent malic enzymes provide mitochondrial NADH for electron transport or cytosolic NADH for reductive power for other metabolic pathways (9)....
Arginase (CARI) gene expression in Saccharomyces cerevisiae is induced by arginine. The 5' regulatory region of CAR) contains four separable regulatory elements-two inducer-independent upstream activation sequences (UASs) (UASCl and UASC2), an inducer-dependent UAS (UASJ), and an upstream repression sequence (URSI) which negatively regulates CA4R and many other yeast genes. Here we demonstrate that three homologous DNA sequences originally reported to be present in the inducer-responsive UASI are in fact three exchangeable elements (UASI.A, UASI-B, and UASI.C). Although two of these elements, either the same or different ones, are required for transcriptional activation to occur, all three are required for maximal levels of induction. The elements operate in all orientations relative to one another and to the TATA sequence. All three UAS, elements bind protein(s); protein binding does not require arginine or overproduction of any of the putative arginine pathway regulatory proteins. The UASI-protein complex was also observed even when extracts were derived from arg80/argRI or arg81/argRll deletion mutants. Similar sequences situated upstream ofARG5,6 andARG3 and reported to negatively regulate their expression are able to functionally substitute for the C4RI UASJ elements and mediate reporter gene expression. CARI (arginase) gene expression in Saccharomyces cere-visiae has been studied as a model of biochemical mechanisms underlying eucaryotic transcription and the cis-and trans-acting factors by which it is regulated. CARI mRNA production is induced to a high level when arginine, the native inducer, is added to the culture medium (1, 5, 17-20, 27, 36). High-level CARI mRNA production is not observed, however, when a readily catabolized nitrogen source such as asparagine or glutamine is present (1,3,5,(18)(19)(20)27); i.e., CARI expression exhibits apparent sensitivity to nitrogen catabolite repression (NCR) (1,3,5,(18)(19)(20)27). CARI NCR sensitivity was generally accepted to result from a discrete transcriptional control process. However, it has recently been shown that CARI sensitivity to NCR occurs via NCRmediated inducer exclusion (4).Dissection of the CAR1 5'-flanking region to identify the sequences required for transcriptional activation and its regulation revealed four separate elements (11-13, 15, 22, 28-31). The first element identified was URS1, a repressorbinding site that functions not only in CARI but also in many other genes, e.g., those participating in meiosis, mating type control, heat shock response, carbon metabolism, inositol metabolism, and oxidative metabolism (2, 9, 10, 14, 15, 22, 28-31, 35, 37). The protein that binds to this site has been purified, and the genes encoding it have been cloned and sequenced (14, 14a). The CARI promoter also contains three discrete upstream activation sequences (UASs) (11-13, 28, 31, 33 currently under study. Thus far, three RAP1 sites and one ABF1 site have been identified (12, 13). However, additional evidence suggests that one or more sites rem...
A mutant malic enzyme gene, mae2-, was cloned from a strain of Schizosaccharomyces pombe that displayed almost no malic enzyme activity. Sequence analysis revealed only one codon-altering mutation, a guanine to adenine at nucleotide 1331, changing the glycine residue at position 444 to an aspartate residue. Gly-444 is located in Region H, previously identified as one of eight highly conserved regions in malic enzymes. We found that Gly-444 is absolutely conserved in 27 malic enzymes from various prokaryotic and eukaryotic sources, as well as in three bacterial malolactic enzymes investigated. The evolutionary conservation of Gly-444 suggests that this residue is important for enzymatic function.
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