A single-strand-specific nuclease from rye germ (Rn nuclease 1) was used for secondary and tertiary structure investigations of tRNAs with large variable arms (class 11 tRNAs). We have studied the structure in solution of two recently sequenced tRNALeU: yeast tRNALeu(nCm5UmAA) and bovine tRNALeu(XmAA) as well as yeast tRNALeU(UAG), tRNALeu(m5CAA) and tRNASer(IGA). The latter is the only tRNA with a long variable arm for which the secondary and tertiary structure has already been studied by use of chemical probes and computer modelling. The data obtained in this work showed that the general model of class 11 tRNAs proposed by others for tRNAser can be extended to tRNAsLeu as well. However interesting differences in the structure of tRNAsLeU versus tRNASer(IGA) were also noticed. The main difference was observed in the accessibility of the variable loops to nucleolytic attack of Rn nuclease I: variable loops of all studied tRNALeU species were cut by Rn nuclease 1, while that of yeast tRNASer(IGA) was not. This could be due to differences in stability of the variable arms and the lengths of their loops which are 3 and 4 nucleotides in tRNAser(IGA) and tRNAsLeu respectively.

A single-strand-specific nuclease from rye germ (Rn nuclease I) was characterized as a tool for secondary and tertiary structure investigation of RNAs. To test the procedure, yeast tRNA(Phe) and tRNA(Asp) for which the tertiary structures are known, as well as the 3'-half of tRNA(Asp) were used as substrates. In tRNA(Phe) the nuclease introduced main primary cuts at positions U33 and A35 of the anticodon loop and G18 and G19 of the D loop. No primary cuts were observed within the double stranded stems. In tRNA(Asp) the main cuts occurred at positions U33, G34, U35, C36 of the anticodon loop and G18 and C20:1 positions in the D loop. No cuts were observed in the T loop in intact tRNA(Asp) but strong primary cleavages occurred at positions psi 55, C56, A57 within that loop in the absence of the tertiary interactions between T and D loops (use of 3'-half tRNA(Asp)). These results show that Rn nuclease I is specific for exposed single-stranded regions.