Loops in Viroids

The structure of the potato spindle tuber viroid (PSTV) was investigated by binding of specific tRNAs to the molecule. Instead of oligonucleotides tRNAs had to be used for those studies because of their higher binding constants and the easier methods of monitoring the extent of binding. Complex formation was detected by analytical ultracentrifugation and by optical titrations. Buffer conditions were 0.05 M sodium cacodylate, 0.01 M MgCl2, pH 6.8 in the optical titrations, and 0.1 M NaCl was added in the ultracentrifugation experiments. From sedimentation analysis at 10°C following results were obtained: tRNALys from Escherichia coli, tRNAphe, tRNAAsp, and tRNASer from yeast bind to PSTV with binding constants in the order of 105 M−1. No binding could be detected with tRNAfMet and tRNA1Val from E. coli. In spectrophotometric titrations with tRNA2Glu and tRNALys from E. coli the decrease in the absorption of mnm5s2U at 300 nm due to the binding was observed. 1 mol tRNA2Glu/mol PSTV binds with a constant of 5.3 × 105 M−1 at 10°C; 3 mol tRNALys/mol PSTV bind with an average constant of 7.0 × 104 M−1 at 20°C. In fluorimetric titrations with tRNAphe from yeast the quenching of the fluorescence intensity of the wye base due to the binding was monitored. 2 mol tRNAphe/mol PSTV bind with an average constant of 7.5 × 105M−1 at 10°C. From the experimental results the following conclusions can be drawn. The anticodons of several specific tRNAs bind to complementary sequences in certain loop regions of PSTV. The loop regions determined by this method are in accordance with the nucleotide sequence and the secondary structure model of PSTV as published recently [2,4]. The bases in the internal loops must have a sufficient degree of flexibility to assume a spatial arrangement which is optimal for base-pairing with other RNAs. The free accessibility of loops distributed over the entire PSTV molecule clearly demonstrates the absence of tertiary structure folding in PSTV.