Binding studies on peptide-oligonucleotide complex: intercalation of tryptophan in GC-rich region of c-myc gene.
Transcriptional regulation of the c-myc gene is essential for normal cellular proliferation; differentiation and overexpression of c-myc is associated with several human cancers. C-myc gene, particularly exon 1, which contains the conserved P1 and P2 promoter regions, has been a potential target for the intercalating drugs in chemotherapy. We have chosen a 21-mer GC-rich oligonucleotide sequence starting from 2281 to 2302 of human c-myc gene located 26 base pair upstream of P1 promoter and partially overlapping with the TATA box of P1. In this paper, we have studied the interaction of a tetrapeptide, KWGK-otBut, with duplex of the above 21-mer sequence under low-salt conditions using UV-Vis absorption, UV melting, fluorescence and circular dichroic (CD) spectroscopy. From the fluorescence quenching data, we determined the two binding constants, K1 (involving only electrostatic interactions) and K2 (involving intercalation), for the formation of (PN)1 and (PN)2 of the two-step mechanism previously established by us. Significant changes were observed in the UV difference absorption spectra and CD spectra of both KWGK and 21-mer duplex upon complex formation even at a very low peptide to nucleotide (P/N) ratios. These spectral changes accompanied by a high value of K2 (=5.13) suggest a strong binding of KWGK involving intercalation of the tryptophan in 21-mer duplex. Based on the above data along with changes observed in deltaH, deltaS and deltaG and increase in melting temperature (by about 8 degrees C) of the 21-mer duplex in presence of KWGK, we propose a model for intercalation of tryptophan of in GC-rich region of c-myc gene. Present observations may be explored in understanding the role of intercalation in protein-nucleic acid interactions in c-myc expression and these results could also help in designing oligopeptides or other low molecular weight ligands to modulate gene expression.
Jain
Akanchha Aklank
AA
Department of Biochemistry, All India Institute of Medical Science, New Delhi 110029, India.
Rajeswari
Moganty R
MR
D002942:Circular Dichroism; D020862:GC Rich Sequence; D016259:Genes, myc; D006358:Hot Temperature; D009841:Oligonucleotides / Q000737:chemistry*; D009842:Oligopeptides / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry*
D009841:Oligonucleotides; D009842:Oligopeptides; D014364:Tryptophan