Multiple determinants within iron-responsive elements dictate iron regulatory protein binding and regulatory hierarchy
Abstract
Iron regulatory proteins (IRPs) are iron-regulated RNA binding proteins that, along with iron-responsive elements (IREs), control the translation of a diverse set of mRNA with 5′ IRE. Dysregulation of IRP action causes disease with etiology that may reflect differential control of IRE-containing mRNA. IREs are defined by a conserved stem–loop structure including a midstem bulge at C8 and a terminal CAGUGH sequence that forms an AGU pseudo-triloop and N19 bulge. C8 and the pseudo-triloop nucleotides make the majority of the 22 identified bonds with IRP1. We show that IRP1 binds 5′ IREs in a hierarchy extending over a ninefold range of affinities that encompasses changes in IRE binding affinity observed with human L-ferritin IRE mutants. The limits of this IRE binding hierarchy are predicted to arise due to small differences in binding energy (e.g., equivalent to one H-bond). We demonstrate that multiple regions of the IRE stem not predicted to contact IRP1 help establish the binding hierarchy with the sequence and structure of the C8 region displaying a major role. In contrast, base-pairing and stacking in the upper stem region proximal to the terminal loop had a minor role. Unexpectedly, an N20 bulge compensated for the lack of an N19 bulge, suggesting the existence of novel IREs. Taken together, we suggest that a regulatory binding hierarchy is established through the impact of the IRE stem on the strength, not the number, of bonds between C8 or pseudo-triloop nucleotides and IRP1 or through their impact on an induced fit mechanism of binding.
Keywords
- iron regulatory protein
- iron-responsive element
- RNA binding protein
- RNA–protein interactions
- iron metabolism
- aconitase
Footnotes
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Reprint requests to: Richard S. Eisenstein, Department of Nutritional Sciences, University of Wisconsin, 1415 Linden Drive, Madison, WI 53706, USA; e-mail: eisenste{at}nutrisci.wisc.edu; fax: (608) 262-5860.
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Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.1857210.
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- Received July 31, 2009.
- Accepted October 5, 2009.
- Copyright © 2010 RNA Society