Abstracts Biological Sciences

Transcriptional regulation of one-carbon metabolism genes of Saccharomyces cerevisiae

by Seung-Pyo Hong

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,…