Identification of key DNA elements involved in promoter recognition by Mxr1p, a master regulator of methanol utilization pathway in Pichia pastoris.
Mxr1p (methanol expression regulator 1) functions as a key regulator of methanol metabolism in the methylotrophic yeast Pichia pastoris. In this study, a recombinant Mxr1p protein containing the N-terminal zinc finger DNA binding domain was overexpressed and purified from E. coli cells and its ability to bind to promoter sequences of AOXI encoding alcohol oxidase was examined. In the AOX1 promoter, Mxr1p binds at six different regions. Deletions encompassing these regions result in a significant decrease in AOXI promoter activity in vivo. Based on the analysis of AOXI promoter sequences, a consensus sequence for Mxr1p binding consisting of a core 5' CYCC 3' motif was identified. When the core CYCC sequence is mutated to CYCA, CYCT or CYCM (M = 5-methylcytosine), Mxr1p binding is abolished. Though Mxr1p is the homologue of Saccharomyces cerevisiae Adr1p transcription factor, it does not bind to Adr1p binding site of S. cerevisiae alcohol dehydrogenase promoter (ADH2UAS1). However, two point mutations convert ADH2UAS1 into an Mxr1p binding site. The identification of key DNA elements involved in promoter recognition by Mxr1p is an important step in understanding its function as a master regulator of the methanol utilization pathway in P. pastoris.
Kranthi
Balla Venkata
BV
Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India.
Kumar
Ritesh
R
Kumar
Nallani Vijay
NV
Rao
Desirazu N
DN
Rangarajan
Pundi N
PN
D000426:Alcohol Dehydrogenase / Q000235:genetics; D000429:Alcohol Oxidoreductases / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D016384:Consensus Sequence; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D016480:Helicobacter pylori / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D000432:Methanol / Q000378:metabolism*; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics* / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D013045:Species Specificity; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
C074682:ADR1 protein, S cerevisiae; D017931:DNA Primers; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D000429:Alcohol Oxidoreductases; C509911:ADH2 protein, S cerevisiae; D000426:Alcohol Dehydrogenase; C019643:alcohol oxidase; D004248:DNA (Cytosine-5-)-Methyltransferases; D000432:Methanol