Structural Investigations of Gene Promoter Region G-quadruplexes and Their Interactions with Proteins

Abstract

The discovery of unique, nonduplex structures in the proximal promoter regions of genes controlling cell growth and proliferation has illuminated a new role for DNA in biology. These non-canonical G-quadruplex structures are formed in loosened guanine-rich promoter regions undergoing torsional stress from processes such as gene transcription. G-quadruplexes, intramolecular four-stranded globular structures most commonly composed of three stacked G-tetrad planes connected by loops, have been found to modulate gene expression by affecting transcription factor binding to gene promoters. Inhibition of transcription factor binding to these regions is likely due to changes in DNA secondary structure and is thought to significantly downregulate expression of certain potent oncogenes. Additionally, certain chaperone-like proteins have been found to induce and stabilize G-quadruplex formation in promoter regions, leading to low gene transcription levels. Thus, structure determination of G-quadruplexes and investigation of protein/G-quadruplex interactions may lead to future structure-based therapeutic design aiming to regulate protein expression. Recently, the proximal promoter region of the Platelet Derived Growth Factor Receptor β (pdgfr-β) gene was found to adopt G-quadruplex structures. Overexpression of PDGFR-β leads to malignancies such as colon cancer and fibrotic disorders. Like previously discovered promoter region quadruplexes, the PDGFR-β structures were found to potentially play a role in controlling gene expression. In the studies described in this dissertation research, the structure of the major, stable Mid-5' PDGFR-β G-quadruplex was determined by NMR. The Mid-5' structure was found to adopt a unique three-tetrad parallel-stranded conformation with a broken-ended strand. Unlike other structures, this G-quadruplex also has a G-triad capping structure protecting the bottom tetrad and two adenine bases protecting the top tetrad. The combination of the broken-ended strand and the two distinctive capping structures provide distinguishing features, which may be exploited for targeted small-molecule drug design. Additionally, the protein nucleolin was formerly found to bind the G-quadruplex in the proximal promoter region of the c-myc gene, which is deregulated in the majority of human cancers. Binding of nucleolin to the c-Myc G-quadruplex was previously shown to decrease gene transcription. In current studies, the interactions between nucleolin and the c-Myc G quadruplex were investigated. Three nucleolin RNA binding domains (RBDs), which are common domains for RNA and DNA binding proteins, are needed to bind the c-Myc G-quadruplex, while all four RBDs from nucleolin (Nuc-1234) are needed to bind the c-Myc G-quadruplex with high affinity. It was that found upon nucleolin binding the c-Myc G-quadruplex maintains its parallel stranded fold, affirmation that nucleolin binds the G-quadruplex conformation as opposed to duplex or single-stranded DNA. Additionally, an unambiguous interaction between K92 of Nuc-1234 makes contacts with the T7 loop region of the c-Myc G-quadruplex, the first mapped contact between a protein and the c-Myc G-quadruplex regulatory element. Currently ongoing studies aim to determine other specific protein/DNA contacts and the complete structure of the nucleolin/c-Myc G-quadruplex complex.