Premium
Identification of Mxr1p‐binding sites in the promoters of genes encoding dihydroxyacetone synthase and peroxin 8 of the methylotrophic yeast Pichia pastoris
Author(s) -
Kranthi Balla Venkata,
Vinod Kumar Hunsur Rajendra,
Rangarajan Pundi N.
Publication year - 2010
Publication title -
yeast
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.923
H-Index - 102
eISSN - 1097-0061
pISSN - 0749-503X
DOI - 10.1002/yea.1766
Subject(s) - pichia pastoris , biology , promoter , gene , dna binding site , zinc finger , microbiology and biotechnology , yeast , genetics , biochemistry , genomic dna , binding site , dna , transcription factor , recombinant dna , gene expression
Abstract Expression of genes involved in methanol metabolism of Pichia pastoris is regulated by Mxr1p, a zinc finger transcription factor. In this study, we studied the target gene specificity of Mxr1p by examining its ability to bind to promoters of genes encoding dihydroxyacetone synthase ( DHAS ) and peroxin 8 ( PEX8 ), since methanol‐inducible expression of these genes is abrogated in mxr1 ‐null mutant strains of P. pastoris . Different regions of DHAS and PEX8 promoter were isolated from P. pastoris genomic DNA and their ability to bind to a recombinant Mxr1p protein containing the N‐terminal 150 amino acids, including the zinc finger DNA‐binding domain, was examined. These studies reveal that Mxr1p specifically binds to promoter regions containing multiple 5′‐CYCC‐3′ sequences, although all DNA sequences containing the 5′‐CYCC‐3′ motif do not qualify as Mxr1p‐binding sites. Key DNA‐binding determinants are present outside 5′‐CYCC‐3′ motif and Mxr1p preferably binds to DNA sequences containing 5′‐CYCCNY‐3′ than those containing 5′‐CYCCNR‐3′ sequences. This study provides new insights into the molecular determinants of target gene specificity of Mxr1p, and the methodology described here can be used for mapping Mxr1p‐binding sites in other methanol‐inducible promoters of P. pastoris . Copyright © 2010 John Wiley & Sons, Ltd.