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Recognition of the initiation codon for protein synthesis in foot-and-mouth disease virus RNA

René Rijnbrand

Department of Molecular Cell Biology, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands

Foot-and-mouth disease virus (FMDV) RNA utilizes two in-frame initiation codons to produce two precursor proteins with identical carboxy termini. The 5' untranslated region (5'UTR) directs the ribosome to internal sequences without the need for a cap structure as used in host mRNAs. The FMDV 5'UTR was cloned upstream of the reporter gene chloramphenicol acetyltransferase (CAT) in order to study the selection of initiation site and to facilitate quantification of the translation products. After in vitro transcription with T7 RNA polymerase and translation in rabbit reticulocyte lysate, the two CAT products, resulting from initiation from the two initiation codons, were quantified. The downstream initiator AUG (AUG_Lb) was selected more efficiently in the wild-type 5'UTR. In truncated RNA, the upstream initiation site (AUG_Lab) was more efficiently utilized than in the wild-type 5'UTR. Protein synthesis initiation factors were added to translation assays to determine whether these factors influenced initiation site selection. Addition of eIF-2 and of eIF-2B changed the selection process for both types of RNA. These factors induced a 2.5-fold higher usage of the upstream AUG_Lab for wild-type and 5'UTR-truncated RNA. A change in mRNA concentration also induced a change in the usage of initiation codons; however, the effect of eIF-2 was measured over a broad range of mRNA concentrations. In conclusion, eIF-2 mediates the recognition of the initiation codon during both cap-dependent and internal ribosome entry site-dependent initiation.

^* Author for correspondence. Fax +31 30 51 36 55. e-mail a.a.m.thomas@biol.ruu.nl

Present address: Department of Virology, Institute of Medical Microbiology, Faculty of Medicine, Leiden University, Postbus 320, 2300 AH Leiden, The Netherlands.

Received 24 July 1995; accepted 7 October 1995.

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