Mapping the cis-acting element(s) in the UTR of BMV RNA2 required for CP-mediated translation inhibition. (A) The 5′ UTR of RNA2, but not RNA3, contains the cis-element required for translation regulation. Agrobacterium cultures expressing reporter RNAs containing combinations of the 5′ and 3′ UTRs of RNA2 and RNA3 were infiltrated into N. benthamiana leaves along with cultures expressing either the empty vector or the BMV CP. GFP was detected by anti-GFP antibody. The slice labeled “Rubisco” is from an SDS-PAGE gel stained with Coomassie blue, which served as a loading control for the Western blotting. (B) Secondary structure of the 5′ UTR of BMV RNA2 from nucleotide 1 to the translation initiation codon. The structure was modeled with the Mfold program (Zuker, 2003). The names of the constructs with deletions within the 5′ UTR of 2GFP2 are shown in bold. (C) Effects of deletions in the RNA2 5′ UTR on GFP accumulation in the absence or presence of exogenously provided CP. (D) Further examination of the effect of B Box deletion on GFP accumulation from 2GFP2.
