Identification of key DNA elements involved in promoter recognition by Mxr1p, a master regulator of methanol utilization pathway in Pichia pastoris

Kranthi, Balla Venkata ; Kumar, Ritesh ; Kumar, Nallani Vijay ; Rao, Desirazu N. ; Rangarajan, Pundi N. (2009) Identification of key DNA elements involved in promoter recognition by Mxr1p, a master regulator of methanol utilization pathway in Pichia pastoris Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms, 1789 (6-8). pp. 460-468. ISSN 1874-9399

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Official URL: http://linkinghub.elsevier.com/retrieve/pii/S18749...

Related URL: http://dx.doi.org/10.1016/j.bbagrm.2009.05.004

Abstract

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.

Item Type:Article
Source:Copyright of this article belongs to Elsevier Science.
Keywords:Alcohol Oxidase; Pichia pastoris; Zinc Finger Protein; Methanol; Transcription; DNase I Foot Printing
ID Code:57894
Deposited On:30 Aug 2011 07:51
Last Modified:05 Jul 2012 08:49

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