High-resolution three-dimensional NMR structure of the KRAS proto-oncogene promoter reveals key features of a G-quadruplex involved in transcriptional regulation

Abstract

Non-canonical base pairing within guanine-rich DNA and RNA sequences can produce G-quartets, whose stacking leads to the formation of a G-quadruplex (G4). G4s can coexist with canonical duplex DNA in the human genome and have been suggested to suppress gene transcription, and much attention has therefore focused on studying G4s in promotor regions of disease-related genes. For example, the human proto-oncogene contains a nuclease-hypersensitive element located upstream of the major transcription start site. The KRAS nuclease-hypersensitive element (NHE) region contains a G-rich element (22RT; 5'-AGGGCGGTGTGGGAATAGGGAA-3') and encompasses a Myc-associated zinc finger-binding site that regulates transcription. The NEH region therefore has been proposed as a target for new drugs that control transcription, which requires detailed knowledge of the NHE structure. In this study, we report a high-resolution NMR structure of the G-rich element within the KRAS NHE. We found that the G-rich element forms a parallel structure with three G-quartets connected by a four-nucleotide loop and two short one-nucleotide double-chain reversal loops. In addition, a thymine bulge is found between G8 and G9. The loops of different lengths and the presence of a bulge between the G-quartets are structural elements that potentially can be targeted by small chemical ligands that would further stabilize the structure and interfere or block transcriptional regulators such as Myc-associated zinc finger from accessing their binding sites on the KRAS promoter. In conclusion, our work suggests a possible new route for the development of anticancer agents that could suppress KRAS expression.

Characterization of clinically used oral antiseptics as quadruplex-binding ligands

“…The structure of the quadruplex in the KRAS promoter is polymorphic, with most conformations containing an atypical single nucleotide bulge ( 18 ). A structure of a G4-forming sequence derived from the KRAS promoter was recently solved by NMR, confirming the proposed structure ( 20 ). Previous studies have provided preliminary evidence that the KRAS G4 can indeed be targeted with small molecules to elicit changes in KRAS expression ( 21–23 ).…”

The regulatory G4 motif of the Kirsten ras (KRAS) gene is sensitive to guanine oxidation: implications on transcription

“…DMS-footprinting and CD experiments showed that 32R folds into a parallel G4 with a thymidine bulge in one strand and a (1/1/11) topology ( 23 , 25 ). Moreover, a truncated portion of 32R, comprising the first four G-runs from the 5′-end, folds into a (1/1/4) G-quadruplex ( 28 , 38 ). We reported that 32R is recognized by several nuclear proteins, including MAZ, PARP-1, Ku70 and hnRNP A1 ( 25 – 27 ).…”

Chemical and structural studies provide a mechanistic basis for recognition of the MYC G-quadruplex

“…Moreover, neither DC-34-binding site in the MYC G4 is obscured by residues from the loop regions, whereas other G4s that we examined have features that make the DC-34 binding sites less accessible. We generated a model of a KRAS G4/DC-34 structure using the KRAS G4 structure (PDB 5I2V) 61 with DC-34 placed in the analogous region compared to its MYC -binding sites. At the 5′ end, the KRAS G4 contains a four-base loop (A14-A15-T16-A17) while the MYC G4 has a one base loop (T19) (Supplementary Fig.…”