title: Chimeric murine interferon regulatory factor-2 (IRF-2) binds to IRF-E (IRF binding element), VREβ (virus response element) but not to VREα1.
abstract: 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 INFβ 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 INFβ and IFNα1 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 VREß , but we could not observe any complex of DNA/GST-IRF-2 with VREα1 . 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 VREß , we used negative control where we observed no binding complex, but we observed complexes with clones IPTG-induced extract. As far as binding on VREα1 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 VREβ but not with VREα1. 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).
authors.affiliation: Recombinant DNA Technology Laboratory, Centre for Biological Science, Central University of Bihar, Patna, India; School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.
authors.forename: Krishna
authors.initials: K
authors.lastname: Prakash
authors.forename: Pardeep
authors.initials: P
authors.lastname: Kumar
authors.forename: Somnath
authors.initials: S
authors.lastname: Mukherjee
authors.forename: P C
authors.initials: PC
authors.lastname: Rath
mesh_terms: D000818:Animals; D019677:Artificial Gene Fusion / Q000379:methods; D004247:DNA / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods; D004926:Escherichia coli; D006801:Humans; D050836:Interferon Regulatory Factor-2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D020218:Response Elements
chemical_list: D050836:Interferon Regulatory Factor-2; C494230:Irf2 protein, mouse; D011993:Recombinant Fusion Proteins; D004247:DNA
keywords: DNA binding domain; gel retardation; interferon; recombinant IRF-2