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Molecular and Cellular Biology, January 2003, p. 687-698, Vol. 23, No. 2
0270-7306/03/$08.00+0     DOI: 10.1128/MCB.23.2.687-698.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Eukaryotic Initiation Factors 4G and 4A Mediate Conformational Changes Downstream of the Initiation Codon of the Encephalomyocarditis Virus Internal Ribosomal Entry Site

Victoria G. Kolupaeva,1 Ivan B. Lomakin,1 Tatyana V. Pestova,1,2 and Christopher U. T. Hellen1*

Department of Microbiology and Immunology, State University of New York Downstate Medical Center, Brooklyn, New York 11203,1 A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, 119899 Moscow, Russia2

Received 10 July 2002/ Returned for modification 9 September 2002/ Accepted 11 October 2002

Initiation of translation of encephalomyocarditis virus mRNA is mediated by an internal ribosome entry site (IRES) comprising structural domains H, I, J-K, and L immediately upstream of the initiation codon AUG at nucleotide 834 (AUG834). Assembly of 48S ribosomal complexes on the IRES requires eukaryotic initiation factor 2 (eIF2), eIF3, eIF4A, and the central domain of eIF4G to which eIF4A binds. Footprinting experiments confirmed that eIF4G binds a three-way helical junction in the J-K domain and showed that it interacts extensively with RNA duplexes in the J-K and L domains. Deletion of apical hairpins in the J and K domains synergistically impaired the binding of eIF4G and IRES function. Directed hydroxyl radical probing, done by using Fe(II) tethered to surface residues in eIF4G's central domain, indicated that it is oriented with its N terminus towards the base of domain J and its C terminus towards the apex. eIF4G recruits eIF4A to a defined location on the IRES, and the eIF4G/eIF4A complex caused localized ATP-independent conformational changes in the eIF4G-binding region of the IRES. This complex also induced more extensive conformational rearrangements at the 3' border of the ribosome binding site that required ATP and active eIF4A. We propose that these conformational changes prepare the region flanking AUG834 for productive binding of the ribosome.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, State University of New York Downstate Medical Center, 450 Clarkson Ave., Brooklyn, NY 11203. Phone: (718) 270-1034. Fax: (718) 270-2656. E-mail: christopher.hellen{at}downstate.edu.

Molecular and Cellular Biology, January 2003, p. 687-698, Vol. 23, No. 2
0022-538X/03/$08.00+0     DOI: 10.1128/MCB.23.2.687-698.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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