title: Interaction of Individual Structural Domains of hnRNP LL with the BCL2 Promoter i-Motif DNA.
abstract: The recently discovered role of the BCL2 (B-cell lymphoma 2 gene) promoter i-motif DNA in modulation of gene expression via interaction with the ribonucleoprotein hnRNP L-like (hnRNP LL) has prompted a more detailed study of the nature of this protein-DNA interaction. The RNA recognition motifs (RRMs) of hnRNP LL were expressed individually, and both RRM1 and RRM2 were found to bind efficiently to the BCL2 i-motif DNA, as well as being critical for transcriptional activation, whereas RRM3-4 bound only weakly to this DNA. Binding was followed by unfolding of the DNA as monitored by changes in the CD spectrum. Mutational analysis of the i-motif DNA revealed that binding involved primarily the lateral loops of the i-motif. The kinetics of binding of the DNA with RRM1 was explored by recording CD spectra at predetermined times following admixture of the protein and DNA. The change in molar ellipticity was readily apparent after 30 s and largely complete within 1 min. A more detailed view of protein-DNA interaction was obtained by introducing the fluorescence donor 6-CNTrp in RRM1 at position 137, and the acceptor 4-aminobenzo[g]quinazoline-2-one (Cf) in lieu of cytidine22 in the i-motif DNA. The course of binding of the two species was monitored by FRET, which reflected a steady increase in energy transfer over a period of several minutes. The FRET signal could be diminished by the further addition of (unlabeled) RRM2, no doubt reflecting competition for binding to the i-motif DNA. These experiments using the individual RRM domains from hnRNP LL confirm the role of this transcription factor in activation of BCL2 transcription via the i-motif in the promoter element.
authors.affiliation: Biodesign Center for BioEnergetics, and School of Molecular Sciences, Arizona State University , Tempe, Arizona 85287, United States.
authors.forename: Basab
authors.initials: B
authors.lastname: Roy
authors.affiliation: Biodesign Center for BioEnergetics, and School of Molecular Sciences, Arizona State University , Tempe, Arizona 85287, United States.
authors.forename: Poulami
authors.initials: P
authors.lastname: Talukder
authors.affiliation: College of Pharmacy, University of Arizona , Tucson, Arizona 85721, United States.
authors.forename: Hyun-Jin
authors.initials: HJ
authors.lastname: Kang
authors.affiliation: Biodesign Center for BioEnergetics, and School of Molecular Sciences, Arizona State University , Tempe, Arizona 85287, United States.
authors.forename: Shujian S
authors.initials: SS
authors.lastname: Tsuen
authors.affiliation: Biodesign Center for BioEnergetics, and School of Molecular Sciences, Arizona State University , Tempe, Arizona 85287, United States.
authors.forename: Mohammad P
authors.initials: MP
authors.lastname: Alam
authors.affiliation: College of Pharmacy, University of Arizona , Tucson, Arizona 85721, United States.|Arizona Cancer Center and BIO5 Institute, University of Arizona , Tucson, Arizona 85721, United States.
authors.forename: Laurence H
authors.initials: LH
authors.lastname: Hurley
authors.affiliation: Biodesign Center for BioEnergetics, and School of Molecular Sciences, Arizona State University , Tempe, Arizona 85287, United States.
authors.forename: Sidney M
authors.initials: SM
authors.lastname: Hecht
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D034441:Heterogeneous-Nuclear Ribonucleoproteins / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D059372:Nucleotide Motifs; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D000072417:Protein Domains; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics*
chemical_list: D034441:Heterogeneous-Nuclear Ribonucleoproteins; D019253:Proto-Oncogene Proteins c-bcl-2; D004247:DNA