Isolation and functional characterization of Spirulina D6D gene promoter: Role of a putative GntR transcription factor in transcriptional regulation of D6D gene expression

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Author list: Subudhi S., Kurdrid P., Hongsthong A., Sirijuntarut M., Cheevadhanarak S., Tanticharoen M.

Publisher: Elsevier

Publication year: 2008

Journal: Biochemical and Biophysical Research Communications (0006-291X)

Volume number: 365

Issue number: 4

Start page: 643

End page: 649

Number of pages: 7

ISSN: 0006-291X

URL: https://www.scopus.com/inward/record.uri?eid=2-s2.0-37049036826&doi=10.1016%2fj.bbrc.2007.11.018&partnerID=40&md5=9e9d6cc293ce5c7ef9592741fe1aee47

Languages: English-Great Britain (EN-GB)

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Abstract

Δ6-Desaturase (D6D) is a key enzyme that catalyzes the synthesis of γ-linolenic acid (GLA), an essential polyunsaturated fatty acid. We report here the isolation and first functional characterization of the D6D gene promoter from Spirulina platensis C1. Functional analysis of this isolated promoter showed that the Spirulina promoter was functional in Escherichia coli. Site-specific mutation studies demonstrated that the -10 sequence (TATAAT), located at -33 bp relative to the translation start site, was essential for D6D promoter function. Temperature responsive deletion analysis studies identified the minimal core promoter within the region -51 to +1, which was sufficient for basal D6D promoter activity, and several cold-shock responsive cis-acting elements with activating and repressing activity. Electrophoretic mobility shift assay and LC-MS/MS studies demonstrated that an 'AT-rich Inverted Repeat' (-192 to -164) served as a target-binding site for a transcriptional regulator (probably a member of the GntR family) from Spirulina. Western blot analysis studies revealed that the DNA-binding transcriptional regulator underwent phosphorylation after a temperature downshift and possibly associated with transcriptional regulation of D6D gene expression. Taken together, our results suggest complex regulation of D6D gene expression in Spirulina. © 2007 Elsevier Inc. All rights reserved.

Keywords

Gene regulation, Promoter, Regulatory element, transcription factor