Nucleic Acids Research, 1992, Vol. 20, No. 17 4631-4638
© 1992
MOLECULAR BIOLOGY |
Identification of the motifs within the tobacco mosaic virus 5'-leader responsible for enhancing translation
Department of Biochemistry, University of California Riverside, CA 92521-0129 USA 1Department of Biological Sciences, Stanford University Stanford, CA 94305-5020, USA
Received March 27, 1992. Revised August 4, 1992. Accepted August 4, 1992.
The leader (called 
) of tobacco mosaic virus RNA enhances translation in both eukaryotes and prokaryotes. Although little secondary structure is predicted to exist within , the primary sequence of the 68 base leader is highly organized. Three copies of an eight base direct repeat and a (CAA)n region represent the two motifs found in the leaders of many TMV strains, and together these comprise 72% of . In previous deletion studies, no mutants exhibited loss-of-function, suggesting that functional redundancy exists within . We report here that a more comprehensive deletion analysis identified the motifs involved in translational enhancement. In a separate approach, oligonucleotides containing the sequence of each motif were used to construct leaders that varied in the number and configuration of the motifs. ß-Glucuronidase mRNA constructs containing these mutant leaders were synthesized in vitro and their translational efficiency measured in vivo following mRNA delivery to carrot protoplasts via electroporation. A combination of one copy of the 8 base direct repeat and a 25 base (CAA)n region was identified as the core regulatory element, although the (CAA)n motif is more critical. Two copies of the (CAA)n region are sufficient to confer a high level of enhancement and a leader composed of multiple copies of the direct repeat is moderately enhancing. Thus, these two motifs are functionally redundant.
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