From an electrophoretic mobility shift assay to isolated transcription factors: a fast genomic-proteomic approach
1 Gene Technology, Department of Gene Technology and Applied Biochemistry, Institute of Chemical Engineering, TU Wien, Getreidemarkt 9/166/5/2, A-1060 Wien, Austria
2 Department of Genetics and Morphology, Institute of Biological Sciences, University of Brasilia, Campus Universitário Darcy Ribeiro, ICC SuL, AT-098, 70.910-900, Brasília-DF, Brazil
3 Research Institute of Molecular Pathology, Vienna Biocenter, Dr. Bohrgasse 7, A-1030 Vienna, Austria
BMC Genomics 2010, 11:644 doi:10.1186/1471-2164-11-644Published: 18 November 2010
Hypocrea jecorina (anamorph Trichoderma reesei) is a filamentous ascomycete of industrial importance due to its hydrolases (e.g., xylanases and cellulases). The regulation of gene expression can influence the composition of the hydrolase cocktail, and thus, transcription factors are a major target of current research. Here, we design an approach for identifying a repressor of a xylanase-encoding gene.
We used streptavidin affinity chromatography to isolate the Xylanase promoter-binding protein 1 (Xpp1). The optimal conditions and templates for the chromatography step were chosen according to the results of an electrophoretic mobility shift assay performed under repressing conditions, which yielded a DNA-protein complex specific to the AGAA-box (the previously identified, tetranucleotide cis-acting element). After isolating AGAA-box binding proteins, the eluted proteins were identified with Nano-HPLC/tandem MS-coupled detection. We compared the identified peptides to sequences in the H. jecorina genome and predicted in silico the function and DNA-binding ability of the identified proteins. With the results from these analyses, we eliminated all but three candidate proteins. We verified the transcription of these candidates and tested their ability to specifically bind the AGAA-box. In the end, only one candidate protein remained. We generated this protein with in vitro translation and used an EMSA to demonstrate the existence of an AGAA-box-specific protein-DNA complex. We found that the expression of this gene is elevated under repressing conditions relative to de-repressing or inducing conditions.
We identified a putative transcription factor that is potentially involved in repressing xylanase 2 expression. We also identified two additional potential regulatory proteins that bind to the xyn2 promoter. Thus, we succeeded in identifying novel, putative transcription factors for the regulation of xylanase expression in H. jecorina.