Effects of sequence elements in the potato virus X RNA 5' non-translated alpha beta-leader on its translation enhancing activity

The 5' non-translated alpha beta-leader sequence of potato virus X RNA consists of two regions: the alpha sequence (41 nucleotides with no G) and the beta sequence (42 nucleotides upstream from AUG). The alpha beta-leader has been shown to enhance strongly the expression of adjacent genes in chimeric mRNAs. This phenomenon has been postulated to be due to the unpaired conformation of the 5'-terminal 30 nucleotides and/or to the presence within the alpha region of the CCACC pentanucleotide complementary to the 3'-terminal conserved structure of 18S rRNA. Different derivatives of alpha beta-leader have been constructed for use in determining the contribution of separate elements of the alpha beta sequence to translational enhancement. It was found that deletion of the alpha sequence large fragment which was supposed to be unfolded did not reduce the delta alpha beta-leader enhancement activity. Moreover, translational enhancement was greater for this derivative. Deletion of the beta sequence resulted in a considerable increase in activity of the alpha-leader showing that the beta region was dispensable for translation. Disruption or 'masking' of CCACC led to inactivation of the alpha beta-leader as a translational enhancer. Thus, we identified the CCACC pentanucleotide as the primary motif responsible for the translation enhancing ability of alpha beta-leader.