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J. Biol. Chem., Vol. 283, Issue 27, 19011-19025, July 4, 2008

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A Small Stem Loop Element Directs Internal Initiation of the URE2 Internal Ribosome Entry Site in Saccharomyces cerevisiae^*

Lucas C. Reineke, Anton A. Komar, Mark G. Caprara^¶, and William C. Merrick¹

From the Department of Biochemistry and ^¶Center for RNA Molecular Biology, Case Western Reserve University, Cleveland, School of Medicine, Ohio 44106 and the Biological, Geological, and Environmental Science Department, Cleveland State University, Cleveland, Ohio 44115

Internal initiation of translation is the process of beginning protein synthesis independent of the m⁷G cap structure at the 5'-end of an mRNA molecule. We have previously shown that the URE2 mRNA in the yeast Saccharomyces cerevisiae contains an internal ribosome entry site (IRES) whose activity is suppressed by eukaryotic initiation factor 2A (eIF2A; YGR054W). In this study, the minimal sequence required to efficiently direct internal initiation was determined using a system that abrogates cap-dependent scanning of the 40 S ribosomal subunit in both wild-type and eIF2A knock-out cells. Subsequently, secondary structural elements within the minimal sequence were determined by probing with RNases T1 and V1 and the small molecule diethylpyrocarbonate. It was found that the URE2 minimal IRES comprises a 104 nucleotide A-rich stem loop element encompassing the internal AUG codon. Interestingly, the internal AUG seems to be involved in base-pairing interactions that would theoretically hamper its ability to interact with incoming initiator tRNA molecules. Furthermore, none of the truncations used to identify the minimal IRES element were capable of abrogating the suppressive effect of eIF2A. Our data provide the first insight into the RNA structural requirements of the yeast translational machinery for cap-independent initiation of protein synthesis.

Received for publication, April 23, 2008

^* This work was supported, in whole or in part, by National Institutes of Health Grants GM-68079, GM-62853, and T32 GM-08056. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Biochemistry, School of Medicine, Case Western Reserve University, 10900 Euclid Ave., Cleveland, OH 44106-4935. Tel.: 216-368-3578; Fax: 216-368-3419; E-mail: wcm2{at}case.edu.

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