The coat protein gene in RNA 3 of alfalfa mosaic
virus (AMV; genus Alfamovirus, family Bromoviridae)
is translated from the subgenomic RNA 4. Analysis of the
subgenomic promoter (sgp) in minus-strand RNA 3 showed
that a sequence of 37 nt upstream of the RNA 4 start site
(nt +1) was sufficient for full sgp activity in an in vitro
assay with the purified viral RNA-dependent RNA-polymerase
(RdRp). The sequence of nt −6 to −29 could
be folded into a potential hairpin structure with a loop
represented by nt −16, −17, and −18,
and a bulge involving nt −23. By introducing mutations
that disrupted base pairing and compensatory mutations
that restored base pairing, it was shown that base pairing
in the top half of the putative stem (between the loop
and bulge) was essential for sgp activity, whereas base
pairing in the bottom half of the stem was less stringently
required. Deletion of the bulged residue A-23 or mutation
of this residue into a C strongly reduced sgp activity,
but mutation of A-23 into U or G had little effect on sgp
activity. Mutation of loop residues A-16 and A-17 affected
sgp activity, whereas mutation of U-18 did not. Using RNA
templates corresponding to the sgp of brome mosaic virus
(BMV; genus Bromovirus, family Bromoviridae)
and purified BMV RdRp, evidence was obtained indicating
that also in BMV RNA a triloop hairpin structure is required
for sgp activity.