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Chimeric murine interferon regulatory factor‐2 (IRF‐2) binds to IRF‐E (IRF binding element), VRE β (virus response element) but not to VRE α1
Author(s) -
Prakash Krishna,
Kumar Pardeep,
Mukherjee Somnath,
Rath P.C.
Publication year - 2014
Publication title -
cell biochemistry and function
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.933
H-Index - 61
eISSN - 1099-0844
pISSN - 0263-6484
DOI - 10.1002/cbf.3050
Subject(s) - electrophoretic mobility shift assay , microbiology and biotechnology , biology , transcription factor , interferon regulatory factors , dna , binding site , gene , dna binding protein , fusion protein , lac operon , gene expression , recombinant dna , genetics
Interferon regulatory factor‐2 (IRF‐2) is a multifunctional transcription factor having gene activation, repression and synergistic effect in conjunction with IRF‐1. IRF‐2 is also involved in type I IFN signalling by repressing INF β gene. So far, the molecular mechanism of its DNA binding activity remains elusive. We have carried out molecular sub‐cloning, expression and electrophoretically mobility shift assay study of chimeric murine IRF‐2. Here, we report expression of chimeric murine IRF‐2 as GST‐IRF‐2 fusion protein in Escherichia coli/BL21 cells and demonstrated DNA binding activity by gel retardation technique using radio 32 P‐labelled IRF‐E motif (GAAAGT) 4 , virus response element (VRE) of human INF β and IFN α1 gene. We observed five different masses DNA/GST‐IRF‐2 complexes (1–5) with IRF‐E motif, three different masses DNA/GST‐IRF‐2 complexes (1–3) with VRE ß , but we could not observe any complex of DNA/GST‐IRF‐2 with VRE α1 . The specific binding on IRF‐E motif was confirmed by carrying out 100‐X fold cold competition with 32 P‐labelled IRF‐E motif. In contrast to specific binding on VRE ß , we used negative control where we observed no binding complex, but we observed complexes with clones IPTG‐induced extract. As far as binding on VRE α1 is concerned, we could not observe any complex in negative control as well as in IPTG‐inducible clones extract. Chimeric IRF‐2 binds with IRF‐E motif and VRE β but not with VRE α1. This study is first of its kind and paves the way to understand the differential DNA binding and molecular mechanism of DNA binding activity of the IRF‐2 molecule, which is crucial for its function(s). Copyright © 2014 John Wiley & Sons, Ltd.