EFFECT OF MODIFIED NUCLEOTIDES ON STRUCTURE OF YEAST TRNA(PHE) - COMPARATIVE-STUDIES BY METAL ION-INDUCED HYDROLYSIS AND NUCLEASE MAPPING

Structural differences between native yeast tRNA(Phe), its in vitro tr anscript and the U8G mutant have been investigated using metal ion-ind uced hydrolysis and nuclease digestion. Differences in the solution st ructure of the molecules involve four regions: the D- and T-loops, the variable region and the anticodon loop. Efficiency of the Pb(II); Eu( II)-, Mn(II)- and Mg(II)-induced hydrolysis at the main cleavage sites in the D-loop is significantly reduced for unmodified tRNAs. Moreover , only the in vitro transcripts are susceptible for cleavage in the T- loop and entire anticodon loop. Other changes in the transcript molecu le involve 50-fold enhancement of S-1 nuclease digestion at p36, weak cleavages in the D-loop and lack of some digestion sites in the T-loop . The nuclease V-1 digestion patterns are very similar for studied mol ecules. Changes in the pattern of hydrolysis of the D-loop caused by m utation of the conservative base U8 to G are detected by metal-induced hydrolysis only. Our results indicate clearly that metal ions acid en zymatic probes monitor different features of RNA structure and their c ombined use is highly advantageous in studying subtle structural chang es in tRNA.