Biochimie 2007, in press
Nasiruddin Khan, Anna Aviñó, Romà Tauler, Carlos González, Ramon Eritja, Raimundo Gargallo
The 5’-end of the P1 promoter of the B-cell Lymphoma-2 (bcl-2) gene contains a highly guanine-cytosine-rich region, which has a role in the regulation of bcl-2 transcription. Whereas the guanine-rich region has been the focus of recent studies, little attention has been paid to the cytosine-rich strand. Here we examine the structural transitions of the cytosine-rich sequence by means of acid-base, mole-ratio and melting experiments monitored by molecular absorption, circular dichroism, and NMR spectroscopies. Two intramolecular i-motif structures have been detected in the pH range 2 - 7, with maximal formation at pH 4 and 6, respectively. At pH 7.6 the majority species has been associated with a hairpin involving Watson-Crick base pairs. Upon addition of the quadruplex-interacting ligand TmPyP4, bcl-2c structures at pH 6.1 and 7.6 yield identical interaction species with stoichiometries 1:2 (DNA : ligand) and logarithms of formation constant 12.4 ± 0.2 and 11.7 ± 0.1, respectively. The initial i-motif structure at pH 6.1 is lost upon interaction with TmPyP4.
Keywords
Multivariate Curve Resolution, i-motif, porphyrin
Acid-base titration of bcl-2c sequence. (A) Experimental CD and absorbance data. (B) Resolved concentration profiles for the four optically active species present along the titration. I and II labels denote the species related to i-motif structures. (C) Resolved pure CD spectra. (D) Resolved pure molecular absorption spectra Continuous line: neutral species, dashed line: i-motif structure I, dotted line: i-motif structure II, dashed-dotted line: protonated species.