Dataset Information

Coexistence of two main folded G-quadruplexes within a single G-rich domain in the EGFR promoter.

ABSTRACT: EGFR is an oncogene which codifies for a tyrosine kinase receptor that represents an important target for anticancer therapy. Indeed, several human cancers showed an upregulation of the activity of this protein. The promoter of this gene contains some G-rich domains, thus representing a yet unexplored point of intervention to potentially silence this gene. Here, we explore the conformational equilibria of a 30-nt long sequence located at position -272 (EGFR-272). By merging spectroscopic and electrophoretic analysis performed on the wild-type sequence as well as on a wide panel of related mutants, we were able to prove that in potassium ion containing solution this sequence folds into two main G-quadruplex structures, one parallel and one hybrid. They show comparable thermal stabilities and affinities for the metal ion and, indeed, they are always co-present in solution. The folding process is driven by a hairpin occurring in the domain corresponding to the terminal loop which works as an important stabilizing element for both the identified G-quadruplex arrangements.

SUBMITTER: Greco ML

PROVIDER: S-EPMC5737278 | biostudies-other | 2017 Sep

REPOSITORIES: biostudies-other

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Coexistence of two main folded G-quadruplexes within a single G-rich domain in the EGFR promoter.

Greco Maria L ML Kotar Anita A Rigo Riccardo R Cristofari Camilla C Plavec Janez J Sissi Claudia C

Nucleic acids research 20170901 17

EGFR is an oncogene which codifies for a tyrosine kinase receptor that represents an important target for anticancer therapy. Indeed, several human cancers showed an upregulation of the activity of this protein. The promoter of this gene contains some G-rich domains, thus representing a yet unexplored point of intervention to potentially silence this gene. Here, we explore the conformational equilibria of a 30-nt long sequence located at position -272 (EGFR-272). By merging spectroscopic and ele ...[more]

PMID: 28973461

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Project description:Investigation of i-motif is of high importance to fully understand the biological functions of G quadruplexes in the context of double-stranded DNA. Whereas single-molecule approaches have profiled G quadruplexes from a perspective unavailable by bulk techniques, there is a lack of similar literature on the i-motif in the cytosine (C)-rich region complementary to G quadruplex-forming sequences. Here, we have used laser tweezers to investigate the structures formed in 5'-(TGTCCCCACACCCC)(2), a predominate variant in the insulin-linked polymorphic region (ILPR). We have observed two species with the change in contour length (DeltaL) of 10.4 (+/-0.1) and 5.1 (+/-0.5) nm, respectively. Since DeltaL of 10.4 nm is located within the expected range for an i-motif structure, we assign this species to the i-motif. The formation of the i-motif in the same sequence has been corroborated by bulk experiments such as Br(2) footprinting, circular dichroism, and thermal denaturation. The assignment of the i-motif is further confirmed by decreased formation of this structure (23% to 1.3%) with pH 5.5 --> 7.0, which is a well-established behavior for i-motifs. In contrast to that of the i-motif, the formation of the second species with DeltaL of 5.1 nm remains unchanged (6.1 +/- 1.6%) in the same pH range, implying that pH-sensitive C:CH(+) pairs may not contribute to the structure as significantly as those to the i-motif. Compared to the DeltaG(unfold) of an i-motif (16.0 +/- 0.8 kcal/mol), the decreased free energy in the partially folded structure (DeltaG(unfold) 10.4 +/- 0.7 kcal/mol) may reflect a weakened structure with reduced C:CH(+) pairs. Both DeltaL and DeltaG(unfold) argue for the intermediate nature of the partially folded structure in comparison to the i-motif. In line with this argument, we have directly observed the unfolding of an i-motif through the partially folded structure. The i-motif and the partially folded structure share similar rupture forces of 22-26 pN, which are higher than those that can stall transcription catalyzed by RNA polymerases. This suggests, from a mechanical perspective alone, that either of the structures can stop RNA transcription.

Dataset Information

Coexistence of two main folded G-quadruplexes within a single G-rich domain in the EGFR promoter.

Publications

Coexistence of two main folded G-quadruplexes within a single G-rich domain in the EGFR promoter.

Similar Datasets

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