The solution conformation of two variants of cucumber mosaic virus sat
ellite RNA (CMV satRNA) was analyzed using several enzymatic and chemi
cal probes. Ribonuclease T1 and nuclease SI were used to map unpaired
nucleotides, and nuclease V1 was used to detect double-stranded, or st
acked, bases. Chemical probing with dimethylsulphate and diethylpyroca
rbonate also identified unpaired and unstacked nucleotides, respective
ly. Modified or cleaved positions were identified by direct gel electr
ophoresis of radioactively labeled RNA, or by analysis of DNA sequence
patterns generated by primer-extension with reverse transcriptase. Ad
ditional information was obtained by a gel-fractionation method under
nondenaturing conditions for the identification of base paired fragmen
ts. On these data, a model for the in vitro secondary structure of CMW
satRNA is proposed. Results support the existence of a complex struct
ure with 51% of nucleotides involved in base pairs (40 G:C, 28 G:U, an
d 18 A:U pairs). Several structural elements, numbered I-VI, were defi
ned, and interactions between separate domains are suggested. Comparis
ons of experimental data and a formerly reported secondary structure m
odel for CMV satRNA support the validity of the structure we propose.