Formation of pseudosymmetrical G-quadruplex and i-motif structures in the proximal promoter region of the RET oncogene.
A polypurine (guanine)/polypyrimidine (cytosine)-rich sequence within the proximal promoter region of the human RET oncogene has been shown to be essential for RET basal transcription. Specifically, the G-rich strand within this region consists of five consecutive runs of guanines, which is consistent with the general motif capable of forming intramolecular G-quadruplexes. Here we demonstrate that, in the presence of 100 mM K+, this G-rich strand has the ability to adopt two intramolecular G-quadruplex structures in vitro. Moreover, comparative circular dichroism (CD) and DMS footprinting studies have revealed that the 3'-G-quadruplex structure is a parallel-type intramolecular structure containing three G-tetrads. The G-quadruplex-interactive agents TMPyP4 and telomestatin further stabilize this G-quadruplex structure. In addition, we demonstrate that the complementary C-rich strand forms an i-motif structure in vitro, as shown by CD spectroscopy and chemical footprinting. This 19-mer duplex sequence is predicted to form stable intramolecular G-quadruplex and i-motif species having minimum symmetrical loop sizes of 1:3:1 and 2:3:2, respectively. Together, our results indicate that stable G-quadruplex and i-motif structures can form within the proximal promoter region of the human RET oncogene, suggesting that these secondary structures play an important role in transcriptional regulation of this gene.
Department of Biochemistry and Molecular Biophysics, University of Arizona, Tucson, Arizona 85721, USA.
Kexiao
K
Guo
Alan
A
Pourpak
Kara
K
Beetz-Rogers
Vijay
V
Gokhale
Daekyu
D
Sun
Laurence H
LH
Hurley
D001483:Base Sequence; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry / Q000235:genetics*; D054856:G-Quadruplexes; D006147:Guanine / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic; D051096:Proto-Oncogene Proteins c-ret / Q000235:genetics*
D006147:Guanine; D004247:DNA; D051096:Proto-Oncogene Proteins c-ret