Date of Award

Spring 11-13-2019

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Deborah S. Wuttke

Second Advisor

Robert T. Batey

Third Advisor

Robin D. Dowell

Fourth Advisor

James A. Goodrich

Fifth Advisor

John L. Rinn

Abstract

Regulation of gene expression by transcription factors (TFs) and their co-regulatory molecules forms much of the basis of how cells interact with their environment. Long noncoding RNAs (lncRNAs) are emerging as a new class of co-regulatory factors, exploiting diverse sequence and structure motifs to interact directly with TFs. An outstanding prototype of this model is glucocorticoid receptor (GR), a TF which binds multiple RNAs in vivo and typifies the molecular decoy model—competitive binding between RNA and DNA at the TF DNA-binding domain. For example, GR binds the noncoding RNA Gas5, a tumor suppressor and growth arrest-specific RNA, via its DNA-binding domain (DBD) with functional implications in pro-apoptosis signaling.

Here we report a comprehensive in vitro binding study where we have determined that GR-DBD is a robust structure-specific RNA-binding domain. GR-DBD binds to a diverse range of RNA hairpin motifs, both synthetic and biologically derived, with apparent mid-nanomolar affinity while discriminating against uniform dsRNA. As opposed to dimeric recognition of dsDNA, GR-DBD binds to RNA as a monomer and confers high affinity primarily through electrostatic contacts. GR-DBD adopts a discrete RNA-bound state, as assessed by NMR, distinct from both free and DNA-bound. NMR and alanine mutagenesis suggest a heightened involvement of the C-terminal α-helix of GR-DBD in RNA-binding. RNA competes for binding with dsDNA and occurs in a similar affinity range as dimer binding to the canonical DNA element. Given the prevalence of RNA hairpins within the transcriptome, our findings strongly suggest that many RNAs have potential to impact GR biology.

Available for download on Wednesday, November 13, 2019

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