Premium
A systematic approach to identify STRE‐binding proteins of the gsn glycogen synthase gene promoter in Neurospora crassa
Author(s) -
Freitas Fernanda Zanolli,
Chapeaurouge Alex,
Perales Jonas,
Bertolini Maria Célia
Publication year - 2008
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200700921
Subject(s) - neurospora crassa , electrophoretic mobility shift assay , biochemistry , microbiology and biotechnology , heat shock protein , biology , gene , dna binding protein , chemistry , transcription factor , mutant
The gene encoding glycogen synthase in Neurospora crassa ( gsn ) is transcriptionally down‐regulated when mycelium is exposed to a heat shock from 30 to 45°C. The gsn promoter has one stress response element (STRE) motif that is specifically bound by heat shock activated nuclear proteins. In this work, we used biochemical approaches together with mass spectrometric analysis to identify the proteins that bind to the STRE motif and could participate in the gsn transcription regulation during heat shock. Crude nuclear extract of heat‐shocked mycelium was prepared and fractionated by affinity chromatography. The fractions exhibiting DNA‐binding activity were identified by electrophoretic mobility shift assay (EMSA) using as probe a DNA fragment containing the STRE motif. DNA–protein binding activity was confirmed by Southwestern analysis. The molecular mass (MM) of proteins was estimated by fractionating the crude nuclear extract by SDS‐PAGE followed by EMSA analysis of the proteins corresponding to different MM intervals. Binding activity was detected at the 30–50 MM kDa interval. Fractionation of the crude nuclear proteins by IEF followed by EMSA analysis led to the identification of two active fractions belonging to the p I s intervals 3.54–4.08 and 6.77–7.31. The proteins comprising the MM and p I intervals previously identified were excised from a 2‐DE gel, and subjected to mass spectrometric analysis (MALDI‐TOF/TOF) after tryptic digestion. The proteins were identified by search against the MIPS and MIT N. crassa databases and five promising candidates were identified. Their structural characteristics and putative roles in the gsn transcription regulation are discussed.