Interactions between eIF4AI and its accessory factors eIF4B and eIF4H
- Department of Biochemistry, Howard Hughes Medical Institute, Brandeis University, Waltham, Massachusetts 02453, USA
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↵1 These authors contributed equally to this work.
Abstract
Ribonucleoprotein complexes (RNP) remodeling by DEAD-box proteins is required at all stages of cellular RNA metabolism. These proteins are composed of a core helicase domain lacking sequence specificity; flanking protein sequences or accessory proteins target and affect the core's activity. Here we examined the interaction of eukaryotic initiation factor 4AI (eIF4AI), the founding member of the DEAD-box family, with two accessory factors, eIF4B and eIF4H. We find that eIF4AI forms a stable complex with RNA in the presence of AMPPNP and that eIF4B or eIF4H can add to this complex, also dependent on AMPPNP. For both accessory factors, the minimal stable complex with eIF4AI appears to have 1:1 protein stoichiometry. However, because eIF4B and eIF4H share a common binding site on eIF4AI, their interactions are mutually exclusive. The eIF4AI:eIF4B and eIF4AI:eIF4H complexes have the same RNase resistant footprint as does eIF4AI alone (9–10 nucleotides [nt]). In contrast, in a selective RNA binding experiment, eIF4AI in complex with either eIF4B or eIF4H preferentially bound RNAs much longer than those bound by eIF4AI alone (30–33 versus 17 nt, respectively). The differences between the RNase resistant footprints and the preferred RNA binding site sizes are discussed, and a model is proposed in which eIF4B and eIF4H contribute to RNA affinity of the complex through weak interactions not detectable in structural assays. Our findings mirror and expand on recent biochemical and structural data regarding the interaction of eIF4AI's close relative eIF4AIII with its accessory protein MLN51.
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Footnotes
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↵2 Present address: Department of Biochemistry and Molecular Pharmacology, Howard Hughes Medical Institute, 825 LRB, 364 Plantation Street, University of Massachusetts Medical School, Worcester, MA 01605, USA.
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Reprint requests to: Melissa J. Moore, Department of Biochemistry and Molecular Pharmacology, Howard Hughes Medical Institute, 825 LRB, 364 Plantation Street, University of Massachusetts Medical School, Worcester, MA 01605, USA; e-mail: melissa.moore{at}umassmed.edu; fax: (508) 856-1002.
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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.1049608.
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↵3 The differences in 4AIII's basal helicase activity and its ability to be stimulated by 4B could be the result of two mutations, P210S and R370Q, that were inadvertently introduced into the 4AIII cDNA by PCR cloning in the Li et al. (1999) paper. The version of 4AIII used here does not contain these mutations.
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- Received March 3, 2008.
- Accepted June 12, 2008.
- Copyright © 2008 RNA Society
