CONTROL OF START CODON CHOICE ON A PLANT VIRAL-RNA ENCODING OVERLAPPING GENES

The signals that control initiation of translation in plants are not w ell understood. To dissect some of these signals, we used a plant vira l mRNA on which protein synthesis initiates at two out-of-frame start codons. On the large subgenomic RNA (sgRNA1) of barley yellow dwarf vi rus-PAV serotype, the coat protein (CP) and overlapping 17K open readi ng frames (ORFs) are translated beginning at the first and second AUG codons, respectively. The roles of bases at positions -3 and +4 relati ve to the AUG codons in efficiency of translation initiation were inve stigated by translation of sgRNA1 mutants in a cell-free extract and b y expression of a reporter gene from mutant sgRNA1 leaders in protopla sts. The effects of mutations that disrupted and restored secondary st ructure encompassing the CP AUG independently of, and in combination w ith, changes to bases -3 and +4 were also examined. Partial digestion of the 5' end of the sgRNA1 leader with structure-sensitive nucleases gave products that were consistent with the predicted secondary struct ure. Secondary structure had an overall inhibitory effect on translati on of both ORFs. In general, the ''Kozak ruled'' of start codon prefer ence predominate in determining start codon choice. Unexpectedly, for a given CP AUG sequence context, changes that decreased initiation at the downstream 17K AUG also reduced initiation at the CP AUG. To expla in this observation, we propose a new model in which pausing of the ri bosome at the second AUG allows increased initiation at the first AUG. This detailed analysis of the roles of primary and secondary structur e in controlling translation initiation should be of value for underst anding expression of any plant gene and in the design of artificial co nstructs.