Terrance G. Cooper, Ph.D.
Office: (901) 448-6175
Lab: (901) 448-4662
Office: 801 M.S.B.
Lab: 811 M.S.B.
Laboratory web page
Research Program:
Protein-nucleic acid interactions in the regulation of eukaryotic gene expression. The development and control of complex biological functions through layered regulatory networks is one important way of integrating molecular processes in eukaryotic cells. The study of such sophisticated networks has been greatly facilitated in the yeast Saccharomyces cerevisiae by the genetics that can be so easily performed with this organism and the fact that the nucleotide sequence of its entire genome is now completed. We have selected a network in yeast that is both sufficiently simple to be understood yet complex enough to be interesting and representative of these systems in higher organisms. The network we are investigating senses the quality of a nitrogen source in a yeast cell's environment and controls the expression of many genes in response to it.
Saccharomyces cerevisiae cells meet their growth requirements for nitrogenous compounds by selective scavenging. The physiological purpose of selectivity is to prioritize the utilization of compounds available so that the most readily-used nitrogen sources are exhausted from the environment before poorly-used compounds are bothered with. This selectivity is accomplished by nitrogen catabolite repression (NCR). NCR is the designation given to the molecular events through which transcriptional activation of genes encoding transport and enzyme proteins needed for uptake and degradation of poorly-used nitrogen sources is maintained at a low level when more readily-used nitrogen sources are available. Until recently, three global nitrogen regulatory factors (Glp3p, Ure2p and DAL80p) were thought to be responsible for NCR-sensitive transcription mediated through the cis-acting UASNTR element containing the sequence 5'GATAAG-3' at its core. Gln3p is a positively-acting regulator that binds to UASNTR elements in the 5' flanking sequences of NCR-sensitive genes and is capable of transcriptional activation in a heterologous assay system. Ure2p is a negative regulator of Gln3p function. When excess nitrogen is available to the cell, Ure2p prevents Gln3p from functioning in NCR-sensitive transcription; it has no effect on Gln3p synthesis. When only poor nitrogen sources are available, Ure2p does not prevent Gln3p from functioning. In contrast to Gln3p, DAL80p is a negatively-acting regulator that has been shown to bind to a subset of UASNTR elements designated URSGATAs. According to the one model, DAL80p is thought to compete with Gln3p for binding to UASNTR elements, and thereby down-regulate Gln3p-mediated transcription. It should be emphasized for clarity that DAL80p is not a required participant for the implementation of NCR. All NCR-sensitive genes remain fully sensitive in DAL80 deletions. Hence NCR of gene expression is the lack of these genes' Gln3p-dependent activation when readily-used nitrogen sources are available. DAL80p, on the other hand, functions to influence the level of inducer-independent gene transcription and the basal level transcription of inducer-dependent genes involved in nitrogen catabolism.
We have recently identified a new transcription factor, Gat1p, which is also required for NCR-sensitive gene expression. Gat1p possesses a zinc finger motif homologous to the GATAAG-binding Gln3 and DAL80 proteins and hence may also bind to some UASNTR elements. This putative characteristic remains to be demonstrated experimentally. Expression of GAT1 is regulated in a manner similar to DAL80, i.e. it is NCR-sensitive, Gln3p-dependent, and DAL80p-regulated.
So far we have described only transcriptional activation and its control as a means of permitting the cell to selectively degrade the best nitrogen sources first. What happens when preferred nitrogen sources are exhausted, i.e. how does the cell sense which poor nitrogen sources are present in its environment and how does it respond to their presence? The answer to this question consists of two parts: first the cell recognizes that poor nitrogen sources are available within its environment by actively transporting them into the cell. In keeping with this mode of sensing, nearly all of the transport systems associated with the uptake of a cell's poor nitrogen sources are regulated by NCR, but their production is not dependent upon inducer; DAL5 encoding the allantoate transport system is a good example. The DAL5 promoter contains only UASNTR elements. In other words, when preferred nitrogen sources run out, production of all its poor nitrogen source transport systems is depressed and they operate full tilt permitting the cell to accumulate any nitrogen source it encounters. Second, although the transport systems operate at full tilt when the cell is scavenging, many of the enzymes required to degrade poor nitrogen sources are still not produced at high levels, i.e. the gene encoding them are not highly expressed, until the poor nitrogen source requiring their action is found and begins to accumulate in the cell. These nitrogen sources or metabolites of them serve as inducers of these degradative genes. The inducible DAL7 gene is a good example of this type of regulation. Its promoter contains 3 types of cis-acting elements: a UASNTR element similar to the one in DAL5, a URSGATA that binds DAL80p, and an inducer-responsive element, the upstream inducer sequence, UIS. The opposing positive and negative actings of the Gln3p and DAL80p are structured such that in the absence of inducer, DAL7 expression occurs at a low basal level. When inducer (allophanate) becomes available as a result of degrading urea, DAl802p, which has been shown to bind to the UIS site, and the DAL80p operate in concert with Gln3p to activate DAL7 gene expression.
We are currently studying the biochemical details of the above molecular events and are identifying the proteins that form the sensing apparatus for the NCR regulation, and inducer-dependent expression. Regulatory networks such as the one described above are being used as models of their mammalian counterparts. The GATA-factor family of DNA binding proteins in mammalian cells are responsible for a wide variety of regulatory functions one of the most important of which is the regulation of heamopoetic tissue specific gene expression, (e.g. the globin genes).
Selected Publications
Cox, K.H., A.B. Pinchak, and T.G. Cooper. 1999. Genome-wide transcriptional analysis in S. cerevisiae by mini-array membrane hybridization. Yeast, 15:703-713.
Beeser. A.E., and T.G. Cooper. 1999. The dual specific protein phosphatase Yvh1p acts upstream of the protein kinase Mck1p in prmoting spore development in Saccharomyces cerevisiae. J. Bacteriol., In press.
Rai, R., J.R. Daugherty, T.S. Cunningham, and T.G. Cooper. 1999. Overlapping positive and negative GATA factor binding sites mediate inducible DAL7 gene expression in Saccharomyces cerevisiae. J. Biol. Chem. 274:28026-28034.
Beeser, A.E., and T.G. Cooper. 1999. Control of nitrogen catabolite repression is not affected by the tRNAGln-CUU mutation, which results in constitutive pseudohyphal growth of Saccharomyces cerevisiae. J. Bacteriol. 181:2472-2476.
Park, H.D., S. Scott, R. Rai, R. Dorrington, and T.G. Cooper. 1999. Synergistic operation of the CAR2 (ornithine transaminase promoter elements in Saccharomyces cerevisiae. J. Bacteriol. In Press.
Svetlov, V., and T.G. Cooper. 1998. The Saccharomyces cerevisiae GATA factors Dal80p and Deh1p can form homo- and heterodimeric complexes. J. Bacteriol. 180:5682-5688.
Svetlov, V., and T.G. Cooper. 1998. Efficient PCR-based random mutagenesis of sub-genic (100 bp) DNA fragments. Yeast 14:89-91.
Svetlov, V., and T.G. Cooper. 1997. The minimal transactivation of Saccharomyces cerevisiae Gln3p is localized to 13 amino acids. J. Bacteriol. 179:7644-7652.
Coffman, J.A., and T.G. Cooper. 1997. Nitrogen GATA factors participate in transcriptional regulation of vacuolar protease genes in Saccharomyces cerevisiae. J. Bacteriol. 179:5609-5613.
Coffman, J.A., R. Rai, D.M. Loprete, T.S. Cunningham, V. Svetlov, and T.G. Cooper. 1997. Cross regulation of four GATA factors that control nitrogen catabolite gene expression in Saccharomyces cerevisiae. J. Bacteriol. 179:3416-3429.
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