The glycine decarboxylase complex (GDC) of Succharomyces cerevisiae composed of four subunits (P, H, T and L) and plays an important role in the interconversion of serine and glycine and balancing the one-carbon unit requirements of the cell. It also enables the cell to use glycine as sole nitrogen source. This study was concerned with characterising the molecular mechanism of transcriptional regulation of the GCVgenes encoding the subunits of the GDC. The important findings of this work can be summarised as follows: i) Transcription of the GCV genes are regulated by glycine and rich nitrogen sources, which are mediated by different cis-acting elements. The LPDl gene did not show a glycine response since its transcriptional regulation is distinct from that of the other genes encoding the GDC subunits. ii) Glycine analogues or serine did not affect expression of GCV2, and therefore glycine probably needs to be metabolised to effect the glycine response of the GCV genes. iii) The repression of the GCV2 gene expression by rich nitrogen sources is mediated by a sequence between -227 and -205 of GCV2, and NCR-regulatory mutant studies showed that repression is not directly controlled by the known NCR system. iv) The glycine response of GCV2 is mediated by a motif (the glycine regulatory region; GRR; 5'-CATCN7CTTCTT-3') with CTTCTT at its core. Additional sequence immediately 5' of this motif (between -310 to -289) plays a minor role for the gene's full glycine response. v) The GRR of the GCV genes can mediate the glycine response by either activation or repression, indicating that the transcription factor(s) mediating the glycine response is/are dual-functional in nature. vi) Studies of GCV2 gene expression using different regulatory mutants showed that expression of the gene is further modulated by other transcription factors such as and Baslp which are distinct from the glycine response and possibly involved in setting up the basal expression level. vii) I n vitro studies of the GRR-protein interaction revealed THF affects the affinity of the DNA-binding protein(s) for the GRR. The importance of THF in regulation of the GCV2 gene was also shown in vivo using a foll mutant that is unable to synthesise any folates. THF or a C1-bound derivative of it acts as a ligand for the transcription factor, thus influencing transcription of the GCV genes in the appropriate physiological manner. viii) Using heparin-Sepharose chromatography fractions, four complex formations (complex I to IV) were observed with the GRR. The protein responsible for one of these was separable from the others. EMSA profiles using the GRR of the GCVI and GCV2 genes (in the presence or absence of THF) were very similar, indicating that these genes bind the same proteins and are regulated in a similar manner. ix) Mutation of the CTTCTT motif within the GRR caused significant reduction in in vitro DNA-protein complex formation, however, THF addition overcame this reduction. x) Only complex II formation was observed with a DNA fragment spanning -322 to -295, and THF affected this complex formation. xi) Footprinting analyses of complex I revealed that the binding protein protected the GRR of the GCV2 gene from DNaseI activity. This protein is an excellent candidate for the glycine response regulatory protein. Titration experiments using EMSA showed that this protein can dimerise. A preliminary genome-wide analysis of the S. cerevisiae transcriptome was carried out using miniarray membrane hybridisation. This investigated the global transcriptional changes within the cell in response to the addition of glycine into the medium. Identification of genes related to various cellular processes including onecarbon metabolism gave an insight into the regulation of the cellular metabolic flow, especially that of one-carbon metabolism. The results indicated that: xii) Glycine is transported into mitochondria to be used as substrate for the GDC which (with mitochondria1 SHMT) produces serine that is subsequently utilised for the various one-carbon metabolic pathways, such as methionine synthesis and purine synthesis. xiii) A gene of unknown function (YER183C) which showed homology to the gene for human 5,lO-CH-THF synthetase was identified from gene-array analysis to be upregulated on glycine addition, indicating the protein encoded by this gene may be involved in balancing the metabolic flow between methionine and purine synthesis when THF pools are disturbed by glycine addition. xiv) Addition of glycine to the medium also triggers the expression of other metabolic genes related to amino acid biosynthetic pathways and that of many other genes which are not directly related to one-carbon metabolism. This may be due to prolonged culturing with glycine in the medium resulting in altered expression of genes mediated by one or more secondary factors. These may reflect an adaptive response rather than a direct consequence of glycine induction. On the basis of the above data, a model for the mechanisms regulating glycine response is presented.
Identifer | oai:union.ndltd.org:ADTP/242293 |
Date | January 1999 |
Creators | Hong, Seung-Pyo, School of Biochemistry & Molecular Genetics, UNSW |
Publisher | Awarded by:University of New South Wales. School of Biochemistry and Molecular Genetics |
Source Sets | Australiasian Digital Theses Program |
Language | English |
Detected Language | English |
Rights | Copyright Seung-Pyo Hong, http://unsworks.unsw.edu.au/copyright |
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