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.