ANALYSIS OF THE IN-VITRO SECONDARY STRUCTURE OF CUCUMBER MOSAIC-VIRUSSATELLITE RNA

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.