Effect of Nrg1 Repressor on NTH1 Transcription and Molecular Docking of Nrg1 on NTH1 Promoter

The amount of intracellular trehalose increases in response to environmental stress in yeast (Saccharomyces cerevisiae). When that stress is terminated, the accumulated trehalose rapidly degrades into glucose rapidly. Synthesis of trehalose is fulfilled by the Trehalose Phosphate Synthase (TPS) enzyme complex, whereas the degradation of trehalose is done by the neutral trehalase enzyme. Under different stress conditions, transcription of the NTH1 gene is activated and Stress Response Elements (STRE) are required for this activation. Nrg1 protein can bind promoters including STRE and PDS elements. Because of the presence of three possible Nrg1 repressor binding sites on the NTH1 promoter, the NTH1 gene may be regulated by the Nrg1 repressor. In order to test this hypothesis, Δnrg1 mutant yeast and its isogenic wildtype yeast strain were used to analyze the transcriptional activation of the NTH1 gene under nitrogen starving conditions. Nth1 transcription of the mutant yeast was seven-fold higher than that of the wild-type under growth conditions, and was not changed during nitrogen starvation. The protein-DNA docking analysis also supported the possibility of Nrg1 binding to the NTH1 promoter. These results revealed that NTH1 gene expression is constitutive in the absence of the Nrg1 repressor protein, hence the transcription of NTH1 is repressed by the Nrg1 protein.


Introduction
Saccharomyces cerevisiae regulates its genetic and metabolic processes to adapt itself to newly changing environments as do all living organisms. Changing gene expression levels in response to environmental stresses, except food deficiency, are rapid and transient [1]. S. cerevisiae accumulates α-1,1 trehalose under unfavorable environmental conditions. When these stress conditions ease, yeast cells convert trehalose into glucose and use it as an energy source. In addition to these functions, trehalose also regulates the glycolytic pathway in yeast cells [2][3][4]. The level of trehalose in the cell changes depending on the stress factors and growth stages. Therefore, the genes and proteins involved in the synthesis and degradation of trehalose must be tightly controlled. Synthesis of trehalose is catalyzed by the Trehalose Phosphate Synthase (TPS) enzyme complex that is composed of four subunits (Tps1p, Tps2p, Tps3p, and Tsl3p) [5,6]. Stress-accumulated trehalose degradation is activated by the neutral trehalase enzyme encoded by the NTH1 gene. Neutral trehalase is localized in the cytoplasm and has an optimum pH value of 7.0 [7,8]. The trehalase enzyme, Nth1, regulates trehalose metabolism by keeping intracellular trehalose at a constant level, and regulates the glycolytic pathway by affecting hexokinase activity [6,9].
The NTH1 gene has a 2079 bp long intron-free coding region and is located on chromosome IV as a single copy. Nth1 protein is an 80 kDa protein and consists of 693 amino acids [10,11]. NTH1gene transcription and the enzymatic activity of the Nth1 protein are increased by heat, oxidative, and metal stress, and by nitrogen starvation [12,13,14]. The NTH1 promoter includes STRE (Stress Response Element) (5'-CCCCT-3') sequences which are necessary for transcriptional activation of the NTH1 gene [9,12,15]. The zinc-finger DNA-binding transcription factors, Msn2 and Msn4, bind to STRE sequences and activate gene expression in response to stress [16]. Activity of the Msn2/4 proteins is controlled by the SWI/SNF1 complex, and the rapamycin (TOR) and cAMP/PKA signaling pathways [17,18]. The target of (the TOR) pathway controls subcellular localization of Msn2/4 by regulating its interaction with cytoplasmic anchor protein, Bmh1 [19]. Activity of the TOR complex is regulated depending on the quality of the nitrogen source [20]. Transcription of the NTH1 gene increases under different stress conditions but trehalase activity has not been observed under the same conditions. Whenever stress conditions are eased, Nth1 protein is activated by cAMP-dependent PKA phosphorylation.
The NRG1 gene encodes the transcriptional repressor protein, Nrg1 (Negative Regulator of Glucose-repressed genes) in S. cerevisiae. Nrg1 is a zinc-finger DNAbinding protein like Msn2/4, and localizes in the nucleus, nucleolus, and anti-nucleolar nucleus within the cell [21]. Nrg1 represses the STA1 gene by interacting with the Ssn6-Tup1 corepressor complex in the presence of glucose [22]. Nrg1 plays a role in the repression of DOG2, SUC2, GAL, and GAL10 genes [23,24]. In addition, Nrg-mediated repression of the FLO11 gene is overcome by the activity of Snf1 kinase, which means Nrg1 is working antagonistically with Snf1 kinase [25]. Genome-wide location analysis has revealed the consensus binding sequence of Nrg1 as GGaCCCT [26]. Analysis of 150 gene promoters repressed by Nrg1/2 showed that Nrg1/2 proteins bind to the STRE-like sequences as in Msn2/4. It has also been found that most of these genes are targets of Msn2/4 and are regulated against oxidative, osmotic, and heat stress, nitrogen starvation, and many other stresses. Therefore, in our research, we investigated the effect of Nrg1 protein on NTH1 gene expression under nitrogen starvation conditions. Computational approaches have become important understanding the protein-DNA interactions involved in many important biological processes such as gene regulation. In the last two decades, experimental results have been complemented with numerous computational approaches to predict the three-dimensional (3D) structural model of interacting macromolecules. Proteinprotein and protein-nucleic acid complexes are the most commonly-attempted targets of molecular docking processes [27][28][29][30][31][32]. NTH1 promoter analysis revealed that three Nrg1 binding sites present in the promoter region. Therefore, in our research, we attempted to predict the three-dimensional structural model of the interaction between the zinc-finger domain of Nrg1 protein and Nrg binding sites on the NTH1 promoter.
In this research we investigated the molecular function of Nrg1p in the transcriptional regulation of the NTH1 gene under nitrogen starvation conditions. In order to validate our experimental results and to determine which of the three Nrg1 binding sequences on the NTH1 promoter is involved in transcriptional regulation, we tried to form an in silico binding model of Nrg1 protein to the NTH1 promoter. Our experimental results clearly indicated that transcription of NTH1 is repressed by Nrg1 protein depending on growth conditions, and our computational results showed that the predicted target sequence of the Nrg1 repressor protein on the NTH1 promoter region is the third CCCT box located -349 bp upstream of the transcriptional start site.
Plasmid used in this research, pNL1, was provided by Professor Jean Marie François (Institut National des Sciences Appliquées, Toulouse, France). pNL1 plasmid containing NTH1-lacZ gene fusion was used to quantitate promoter activity of the NTH1 gene in response to varying growth conditions. In this expression vector, the 770bp promoter region, upstream of the translation start site, the NTH1 gene fused in-frame to the lacZ gene. It has been shown that this promoter region contains all of the regulatory sites required for NTH1 gene expression [34]. pNL1 is a Yep353-based expression vector stably maintained in selective growth conditions in S. cerevisiae transformants [35]. Escherichia coli strain, DH5α, was used to amplify both plasmid DNAs.
S. cerevisiae strains were cultured in YPD medium (1% Yeast Extract, 2% Bactopeptone and 2% glucose) for plasmid transformation. The plasmids were transformed into the wild-type and Δnrg1 mutant yeast strains using lithium acetate-polyethylene glycol procedure as described previously [36]. Selection of transformants was done on the yeast synthetic drop-out medium without uracil (YSD w/o URA), supplemented with 2% glucose and grown at 30°C in an incubator to get wellgrown colonies. Yeast colonies were patched to fresh YSD plates and grown for 2-3 days at 30°C. These yeast patches were used in liquid culture inoculations.

Growth Conditions.
To determine the doubling times (dt) and specific growth rates (μ) of the yeast strains, yeast cells were grown in a minimal medium containing 0.17% yeast nitrogen base, 0.5% ammonium sulfate, 2% glucose, and the appropriate amino acids. Optical densities of yeast cultures (OD 600 ) were measured spectrophotometrically every 2 h and used for calculation.
Wild type and Δnrg1 mutant yeast transformants were grown overnight in YSD culture supplemented with 2% glucose at 120 rpm and 30°C to get cultures. Saturated overnight cultures were the same culture and grown to an optical density of 0.8 1.0 (A 600 ) at 30°C with constant shaking. At the end of this growth period, the yeast cultures were divided into two parts, and half was directly used for enzyme assays (non-treated culture). The second part of the culture was harvested and washed twice with sterile distilled wa the resuspended in fresh YSD culture with 0.1% proline instead of ammonium sulfate further incubated 4 h at 30°C. At the end of incubation periods, yeast cells were harvested and used for the measurements of β-galactosidase activities and the trehalose content of the yeast transformants.
Trehalose Assay. Trehalose assays of the transformant yeast cells were determined as described previously [34]. Yeast transformants were removed with ice-cold water, resuspended in 250 µL of 0.25 M Na 2 CO 3 , and boiled for 2 h. 150 µL of 1 M acetic acid and 600 µL of 0.2 M sodium acetate pH 5.2 were added. The cell mixture was incubated in the prese of 3 mU trehalase enzyme (Sigma, T8778) at 37 18 h. Amount of the liberated glucose was determined enzymatically via the glucose oxidase system (GOD-POD assay) using a commercial kit (Fluitest®-GLU, Biocon, Germany) [39 trehalose content of the yeast cells w micrograms of glucose equivalent per milligram mass (µg glucose/mg cell wet weight) of the yeast cells.
In silico Analysis. NTH1 promoter region (1000 bp) and Nrg1 amino acid sequence were obtained from Saccharomyces cerevisiae Genome Database potential Nrg1 binding site on NTH1 determined using the JASPAR CORE data base (ID:MA0347.1) and are given in Figure  DNA fragments, including Nrg1 binding site(s) converted to .pdb format using the

Effect of Nrg1 Repressor on NTH1 Transcription and Molecular
September°C to get saturated yeast aturated overnight cultures were refreshed in same culture and grown to an optical density of 0.8-°C with constant shaking. At the end of period, the yeast cultures were divided into rectly used for enzyme assays treated culture). The second part of the culture was harvested and washed twice with sterile distilled water, the resuspended in fresh YSD culture supplemented % proline instead of ammonium sulfate, and°C . At the end of the incubation periods, yeast cells were harvested and used galactosidase activities and transformants.
galactosidase activity determined as described After completion of incubation, the rvested yeast cells were washed and resuspended in breaking buffer (100 mM Tris HCl mM PMSF). Cells 20 µL of 0.1% SDS and 20 µL Buffer (60 mM Na 2 HPO 4 , 40 mM , 10 mM KCl, 1 mM MgSO 4 , 50 mM βlactosidase units were β-D-galactopyraper milligram of protein in permeabilized yeast cells. Protein conwere determined by Lowry assay and BSA Yeast transformants were galactosidase assays repeated lactosidase units Trehalose assays of the transformant yeast cells were determined as described previously Yeast transformants were removed and washed resuspended in 250 µL of 0.25 M 150 µL of 1 M acetic acid and 600 µL of 0.2 M sodium acetate pH 5.2 were then added. The cell mixture was incubated in the presence ase enzyme (Sigma, T8778) at 37°C for mount of the liberated glucose was determined enzymatically via the glucose oxidase-peroxidase POD assay) using a commercial kit [39]. Determined yeast cells was given as milligram of wet /mg cell wet weight) of the yeast cells.
promoter region (1000 bp) and Nrg1 amino acid sequence were obtained from the Genome Database. parameters [40]. The 3D-DART web the fasta formatted files that included 5'-3' template strand. The an additional restraint file to maintain the DNA conformation during the flexible refinement his case server added 60 bases on ' site of the template strand. The 3D-DART server riven DNA Analysis and Rebuilding Tool) a convenient means of generating custom3D els of DNA with control over local and isualization of the two 'nrg1p_znf1.pdb' and 'nrg1p_znf2.pdb' files was done [41]. UCSF Chimera is a highly extensible program for interactive visualization and analysis of molecular structures and related data. Predicted 3D models of both Nrg1 protein (231 residues) and C 2 H 2 domain (residues between 174 and 226) were created by means of the I-TASSER webserver with default parameters [42,43,44]. I-TASSER (Iterative Threading ASSEmbly Refinement) predicted protein structure and function in a hierarchical approach. It first identified structural templates from the PDB by the multiple threading approach LOMETS, with full-length atomic models constructed by repetitive template-based fragment assembly simulations. I-TASSER predicted the top five final models for both Nrg1 full protein and C 2 H 2 domain according to C-score. Confidence of each model was quantitatively measured by C-score. C-score is typically in the range of -5 to 2, where a C-score of a higher value signifies a model with a higher confidence. For molecular docking, the .pdb files of Nrg1 (full protein and C 2 H 2 domain) obtained from the I-TASSER and the NTH1 promoter fragments (nrg1p_znf1.pdb and nrg1p_znf2.pdb) were uploaded to the NPDock web server with default parameters [45]. NPDock (Nucleic acid-Protein Dock) is a web server for the modeling of RNA-protein and DNA-protein complex structures. UCSF Chimera v.1.13.1 was used for visualization of the NPDock files [41].

Results
Yeast strains used in this research, BY4741 and Y03979 (Δnrg1), were incubated in a minimal medium at 30°C for 48 h. Optical densities of yeast cultures (OD 600 ), measured spectrophotometrically every 2 h, were used for calculation of doubling times (dt) and specific growth rates (μ). Doubling times of Δnrg1 and wildtype yeast cells were determined to be 217 min (3 h 37 min) and 156 min (2 h 36 min), respectively. Specific growth rate of Δnrg1 yeast cells (0.2003 h -1 ) was slower than that of the wild-type (0.2848 h -1 ). In order to determine the metabolic and genetic effects of environmental changes, yeast cells must complete at least one cell cycle. Cell cycles were completed in 3.5 and 2.5 hours in Δnrg1 mutant and wild-type yeast cells, respectively. For this reason, the incubation period performed in the next steps was 4 h for both yeast strains.
Effect of Nrg1 Protein on NTH1 Gene Expression and Trehalose Accumulation. Nrg1 represses some genes involved in carbohydrate metabolism such as STA1, DOG2, SUC2, GAL1, and GAL10 genes [22,23,24]. The NTH1 gene is also involved in carbohydrate metabolism and regulated by glucose level as in STA1 and SUC2 genes. Therefore, NTH1 gene expression may be controlled by Nrg1 protein. NTH1-LacZ gene fusion includes 770 bp of the NTH1 promoter region, which contains STRE sequences and other cis-elements necessary for the regulation of NTH1 gene expression [34]. NTH1 gene expression level and trehalose amount were determined in exponentially growing yeast transformants.
Beta galactosidase enzyme activity in wild-type yeast cells was calculated as 76.8±8.6 Units, and in Δnrg1 mutant yeast cells 555.7 ± 34.2 Units (Figure 2). NTH1 expression in Δnrg1 mutant yeast cells was observed to be about 7x greater than in wild-type yeast strain. The high level of NTH1 transcription in the absence of Nrg1 protein suggested that Nrg1 protein was involved in the repression of NTH1 gene. It is known that Nrg1 protein acts as a negative regulator of carbon-repressed genes in S. cerevisiae yeast cells [24]. Similarly, our results showed that Nrg1 protein was a negative regulator of the NTH1 gene.
In order to determine the effect of Nrg1 protein on trehalose accumulation, trehalose content of the yeast cells was enzymatically hydrolyzed into glucose. Released glucose was then measured. The amount of trehalose was given as micrograms of glucose measured in mgs cell wet weight of the yeast cells. The amount of trehalose measured in the wild-type yeast cells (277.4 ± 57.1 μg glucose/mg cell wet weight) was 2x the amount of trehalose measured in the Δnrg1 mutant yeast cells (100 ± 14,2 μg glucose/mg cell wet weight) (Figure 3). The high NTH1 gene expression in Δnrg1 mutant yeast cells may have resulted in a breakdown of trehalose. However, newly synthesized trehalase enzyme was inactivated. It was activated by PKA-mediated phosphorylation after environmental stress terminated. This means that, the high level of NTH1 transcription was not directly proportional to trehalase activity and could not cause that amount of trehalose accumulation in Δnrg1 mutant yeast cells. The strong probability was that Nrg1 protein may have been the repressor of genes involved in trehalose synthesis. Therefore, TPS1 gene expression in Δnrg1 mutant yeast cells needs to be determined.

Effect of Nitrogen Starvation on NTH1 Gene
Expression and Trehalose Level. Glutamine, glutamate, asparagine and ammonium are good and preferred nitrogen sources for S. cerevisiae yeast cells, while proline and urea are weak and poor nitrogen sources. Expression of genes involved in the uptake and utilization of poor nitrogen sources is repressed in the presence of strong nitrogen sources by activity of the nitrogen catabolite repression mechanism [46,47]. The Tor signaling pathway, which regulates NTH1 gene expression via Msn2/4 transcription factors, is regulated depending on the quantity and quality of nitrogen. We therefore analyzed the effects of poor nitrogen sources on NTH1 gene expression and trehalose accumulation, both in the wild-type and Δnrg1 mutant yeast cells. First, yeast cells were grown to logarithmic stage in the preferred nitrogen source, ammonium, then washed and transferred to a poor nitrogen source, proline. After four-hour incubation, yeast cells were harvested and used for determination of beta galactosidase activity and trehalose content.

. Transcription Levels of Wild Type and Content is Given as Micrograms of Glucose per Milligram of Wet Weight of the Yeast Cells
β-galactosidase activities of wild-type and yeast strains were measured at 626.0 ± 553.6±55.5 Units, respectively, when transferred into the poor nitrogen source (Fig. 2). As shown, transcription increased about 8-fold in yeast strains, while transcription levels much in the Δnrg1 mutant yeast cells under starvation conditions.

Effect of Nrg1 Repressor on NTH1 Transcription and Molecular
September

Mutant Yeast Cells Before and After Nitrogen Starvation. Trehalose Content is Given as Micrograms of Glucose per Milligram of Wet Weight of the Yeast Cells
the growth condition of the low nitrogen signaling and caused like repression of the TOR signaling ression of Tor signaling resulted in localization of Msn2/Msn4 in the nucleus and bound to NTH1 transcription. If we otein is not involved in starvation conditions, due to the occupation STRE by Msn2/4 factors, the similar levels of NTH1 transcription Δnrg1 and wild-type yeast cells can be expect Transcription of NTH1 in Δnrg1 mutant yeast cells d not change before and during starvation meant that Nrg1 repressor was essential for transcription under normal growth conditions, where NTH1 gene expression was repressed.
It has been reported that the accumulation of trehalose starts as a diauxic shift and continues un phase of growth in S. cerevisiae yeast cells Similarly, the trehalase activity is low in phase and begins to increase during the [49]. Also, yeast cells accumulate trehalose in re to stress irrespective of growth phase. The trehalose contents of starved wild-type and Δnrg1 cells were determined to be 656,1 ± 68,4 and 1298 72,4 μg glucose/mg cell wet weight, (Figure 3). It was determined that the am trehalose increased two-fold in wildand 13-fold in Δnrg1 mutant yeast cells after to nitrogen starvation. It was shown that the trehalose level in mutant yeast cells under nitrogen starvation was 2x that of the wild type.

In silico Binding of Nrg1 Protein to NTH1
Computational techniques were used for elucidating a theoretical model of protein-DNA interactions. The three-dimensional structure of protein and sequences must be selected for making a good protein DNA docking model that is close to its native interaction. Our experimental results showed that Nrg1 protein had a negative role in NTH1 transcription assumed that Nrg1 binds to the NTH1 promotor STA1 gene [22]. From this perspective, we determine in silico binding of Nrg1 repressor protein to the NTH1 promoter region and to determine which binding sequence motif was involved in Predicted 3D models of the full Nrg1 protein finger-C 2 H 2 domain were created by means of I TASSER [42,43,44]. I-TASSER predicted the final models for both Nrg1 full protein and C according to C-score. The protein highest C-scores are seen in Figure 4A and protein and C 2 H 2 domain, respectively. Nrg1 protein was used for molecular docking to (nrg1p_znf1.pdb and nrg1p_znf2.pdb) with a default value for RMSD threshold since values up to 10 Å gave the most reasonable results [45]. Within the RMSD threshold values, no simulation was obtained for the first DNA fragment (nrg1p_znf1.pdb). However, the second DNA fragment (nrg1p_znf2.pdb) revealed a reasonable simulation ( Figure 5). The second DNA fragment include Nrg1 binding sites so one or both of them for interaction. In order to determine between atoms of amino acid residues and nucleotides, molecular interaction of the C 2 H 2 domain September 2019 transcription in both yeast cells can be expected. mutant yeast cells did during starvation conditions. That s essential for NTH1 normal growth conditions, where It has been reported that the accumulation of trehalose until the stationary yeast cells [48]. is low in the exponential the stationary phase yeast cells accumulate trehalose in response growth phase. The trehalose Δnrg1 mutant yeast 68,4 and 1298 ± 72,4 μg glucose/mg cell wet weight, respectively ). It was determined that the amount of -type yeast cells mutant yeast cells after exposure shown that the trehalose level in mutant yeast cells under nitrogen starvation was NTH1 Promoter. e used for elucidating a DNA interactions. The al structure of protein and target DNA sequences must be selected for making a good proteinclose to its native ur experimental results showed that Nrg1 transcription. We promotor as in the , we attempted to binding of Nrg1 repressor protein to and to determine which Nrg1 in this interaction. full Nrg1 protein and zincdomain were created by means of I-TASSER predicted the top five final models for both Nrg1 full protein and C 2 H 2 domain protein models having A and 4B for Nrg1 domain, respectively. Nrg1 protein was used for molecular docking to DNA fragments znf2.pdb) using NPDock ault value for RMSD threshold set to 10 Å the most reasonable results Within the RMSD threshold values, no simulation was obtained for the first DNA fragment the second DNA fragment (nrg1p_znf2.pdb) revealed a reasonable simulation The second DNA fragment included two so one or both of them could be used In order to determine close contacts amino acid residues and nucleotides, domain with the second DNA fragment (nrg1p_znf2.pdb) was formed using NPDock with the same parameters contacts between the atoms of nucleotides localized in the determined and 3D structure of each interaction is Figure 6. Results showed the on the non-template strand (5 Amino acid residues in the (T 12 ), histidine (H 15 ) and arginine (R interaction with A 96 , G 98 (or G ( Figure 6). Computational analysis reveal potential binding site of the Nrg1 protein to promoter was localized -349 bp upstream of transcriptional start site.  In our study, it was determined that NTH1 gene fold in Δnrg1 mutant yeast cells under normal growth conditions and did not change in nitrogen starvation. This suggested that Nrg1 required for repression of the NTH1 gene. a negative regulator of glucoserepressible genes, but the binding sequences of Nrg1 and Mig1 proteins on DNA were completely different. While the repression mechanism of Mig1 protein, which dependent phosphorylation, as was clearly revealed, there was no sufficient information about the mechanism of Nrg1 repression. It has been suggested that Nrg1 and Nrg2 proteins may be directly or indirectly targeted by Snf1 kinase [23]. However, the appropriate phosphorylation sites on Nrg1 protein for Snfl kinase have not yet been identified. For this reason, it is thought that the repression mechanism of Nrg1 is different from Mig1 [51].
Mig1 is not sufficient for repression of some glucoserepressible genes. It is known that some genes can be repressed in the absence of Mig1, or that some other genes cannot completely be repressed, even if the Mig1 protein binds to their promoters. More than one repressor protein can bind to a promoter to ensure complete glucose repression [52]. It was shown that the Nrg1 protein specifically bound to two regions in the upstream activation sequence of the STA1 gene; deletion of the NRG1 gene caused an increase in STA1 transcription in the presence of glucose [22]. Nrg1 acted as a DNA-binding repressor and mediated glucose repression of the STA1 gene expression by recruiting the Ssn6-Tup1 complex. Expression of SUC2, GAL1, and GAL10 genes in Δmig1, Δnrg1, and Δmig2 mutant yeast strains was lower than in the Δnrg1Δmig1Δmig2 triplemutant yeast strain [24]. Therefore, as in the STA1 gene, Nrg1 protein may be necessary for 'complete repression' of NTH1 gene expression.
Trehalose synthesis is accomplished by the TPS enzyme complex. Stress-dependent trehalose degradation is carried out by Nth1. Breakdown of trehalose is controlled by a number of transcriptional and posttranslational mechanisms. In our study, the amount of trehalose increased two-fold during nitrogen starvation conditions in the wild-type yeast cells. However, deletion of the NRG1 gene caused a 13-fold increase in NTH1 transcription during nitrogen starvation. This suggested that the Nrg1 protein may also be the repressor of the TPS complex.
Our computational analysis revealed that Nrg1 protein can bind to NTH1 promoter for regulating transcription. NTH1 promoter includes three putative binding site for Nrg1 protein. We showed that the third Nrg1 binding site was localized -349 bp upstream from the transcription initiation site, and is a powerful binding site for Nrg1 protein. A similar molecular approach may be applied to TPS1 promoter.

Conclusion
In conclusion, results of this study show that Nrg1 protein repress the transcription of the NTH1 gene. Nrg1 protein may interact with other repressor proteins and may be involved in the complete repression of the NTH1 gene. Our computational modeling of protein-DNA docking supports the possibility of Nrg1 binding to the NTH1 promoter region. Nrg1 protein may also be involved in the repression of genes involved in TPS complex formation. To support these data, further genetic and biochemical analysis must be conducted in the future using Δmig1, Δmig2, and Δmig1Δmig2 double-mutant yeast strains.