THE PLANT CELL, Vol 5, Issue 6 679-692, Copyright © 1993 by American Society of Plant Biologists
Control of Start Codon Choice on a Plant Viral RNA Encoding Overlapping Genes
S. P. Dinesh-Kumar and W. A. Miller
Plant Pathology Department and Molecular, Cellular, and Developmental Biology Program, 351 Bessey Hall, Iowa State University, Ames, Iowa 50011-1020
The signals that control initiation of translation in plants are not well
understood. To dissect some of these signals, we used a plant viral mRNA on
which protein synthesis initiates at two out-of-frame start codons. On the
large subgenomic RNA (sgRNA1) of barley yellow dwarf virus-PAV serotype,
the coat protein (CP) and overlapping 17K open reading frames (ORFs) are
translated beginning at the first and second AUG codons, respectively. The
roles of bases at positions -3 and +4 relative to the AUG codons in
efficiency of translation initiation were investigated by translation of
sgRNA1 mutants in a cell-free extract and by expression of a reporter gene
from mutant sgRNA1 leaders in protoplasts. The effects of mutations that
disrupted and restored secondary structure encompassing the CP AUG
independently of, and in combination with, changes to bases -3 and +4 were
also examined. Partial digestion of the 5[prime] end of the sgRNA1 leader
with structure-sensitive nucleases gave products that were consistent with
the predicted secondary structure. Secondary structure had an overall
inhibitory effect on translation of both ORFs. In general, the "Kozak
rules" of start codon preference 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 explain this observation, we propose a new model in which
pausing of the ribosome at the second AUG allows increased initiation at
the first AUG. This detailed analysis of the roles of primary and secondary
structure in controlling translation initiation should be of value for
understanding expression of any plant gene and in the design of artificial
constructs.
