Improved procedure for the isolation of a double-strand-specific ribonuclease and its application to structural analysis of various 5S rRNAs and tRNAs

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Abstract
An improved method for the isolation of a double-strand-specific RNase from snake venom is presented. This RNase, called CSV, was used to cleave yeast tRNAPhe and tRNA2Glu and tRNAfMet from Escherichia coli. In addition these RNAs and E. coli tRNAPhe were examined with the single-strand-specific nuclease S1. The results are discussed in terms of the specificity of CSV RNase and the structure of tRNAs. S1 nuclease digestion at increasing temperatures allowed the melting of tertiary and secondary structure to be monitored. 5s rRNA from E. coli, Thermoplasma and the chloroplasts of Spinacia oleracea were digested with CSV and S1. The information these results give on the secondary-structural differences between different classes of 5S rRNA is discussed. Supporting evidence is found for tertiary interactions between hairpin loop c and internal loop d of eubacterial 5S rRNA.