Plant Molecular Biology Reporter pages 347-357 12 (4) 1994 Protocol Plant Expression Cassettes for Enhanced Translational Efficiency M. J. Dowson Day, J. L. Ashurst and R. A. Dixon Nitrogen Fixation Laboratory, University of Sussex, Falmer, Brighton, BN1 9RQ, UK (fax: 44-273-678133) Key Words: tobacco mosaic virus, untranslated leader, plant vector, GUS Abstract: A number of plant expression cassettes have been constructed that are designed to ensure efficient ribosome recruitment, thereby improving transla- tional efficiency. For this purpose the cassettes employ the untranslated leader of the tobacco mosaic virus (TMV) RNA. They also comprise the cauhflower mosaic virus (CaMV) 35 S promoter, with duplicated upstream elements, and the nopaline synthase (nos) terminator. Cloning nests were included 3' to the TMV leader and 5' to the promoter to facilitate the construction of transcriptional or translational fusions and promoter modifications. In both a transient assay system and transgenic tobacco these cassettes were found to facilitate high- expression levels of a cytosolic and plastid-targeted reporter protein (~-glucu- ronidase). Also they enabled the detection of a hitherto very poorly expressed protein. L ow levels of expression of foreign genes in transgenic plants led us to investigate expression levels of a reporter gene (~-glucuronidase: GUS) when different 5'-untranslated viral RNA leaders were introduced into the construction. Of those tested the TMV leader was found to be the most efficient, increasing GUS translational efficiency by 3.5 to 4 fold in transgenic tobacco (Dowson Day et al., 1993). The ability of the TMV leader to enhance translational efficiency may be attributed in part to its length (of 68 nucleotides) (Kozak, 1991) and also to the likelihood that it participates in little or no secondary structure (Sleat et al., 1988). These characters are beheved to facilitate scanning of the 5'- Abbreviations: CaMV, cauliflower mosaic virus; MU, methyl-umbelliferone; nos, gene encoding nopaline synthase; TMV, tobacco mosaic virus. 347