Figure 4 | Scientific Reports

Figure 4

From: ZCF32, a fungus specific Zn(II)2 Cys6 transcription factor, is a repressor of the biofilm development in the human pathogen Candida albicans

Figure 4The alternative text for this image may have been generated using AI.

SELEX revealed the sequence-specific binding of Zcf32 to double-stranded DNA. (A) An electrophoretic mobility shift assay (EMSA) was performed using purified Zcf32 ZFN-MBP fusion protein (with increasing protein concentrations as indicated) and ATP-[γ-32P] labelled 18 bp double-stranded DNA oligonucleotides 1 and 2 (O1- TACCCGATATAGCCGATG and O2- CCGATATAGCCGATGCAT). The mobility shift is illustrated by an asterisk. (B) SELEX revealed that Zcf32 binds to eighteen independent double-stranded DNA sequences. A consensus analysis was carried out for these DNA sequences using MEME analysis tool and a 9 bp long consensus logo was obtained. Below the logo, sequences of the 18 bp long ds DNA oligonucleotides O1, O2 and the variant V1 are shown wherein the consensus region is underlined. In the case of the variant oligonucleotides, the mutated base is highlighted in red colour. (C) An EMSA was carried out with oligonucleotide O1and variant oligonucleotides (V1 to V27) to analyze the critical positions in the binding consensus of Zcf32. The binding ability of recombinant Zcf32 ZFN-MBP protein was analyzed for 27 variant oligonucleotides. Two representative EMSA gels for O1 and variant oligonucleotides V1 to V9 are shown. Asterisk indicates the shift in the mobility of ATP-[γ-32P] labelled ds DNA. The second lane for each oligonucleotide sequence in the EMSA gel indicates the probe (ATP-[γ-32P] labelled ds DNA) control. (D) Summary of the results from the mutational analysis of Zcf32 binding consensus sequence is represented in the form of a bar graph. The intensity of the shifted DNA was measured using Multigauge tool for each reaction carried out. The intensity of the shifted band obtained from variant DNA oligonucleotide was divided by the same of O1 oligonucleotide, thus giving the fraction of protein bound to the particular oligonucleotide sequence. Finally, the data is presented as percent binding of Zcf32 ZFN-MBP to O1and variant oligonucleotides in the form of a bar graph. Mutational analysis with single base pair change at a particular position shows that T, A and C at positions 6, 7 and 9 respectively are critical for the binding of Zcf32 ZFN domain.

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