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.