D. Michalowski et al., EFFECT OF MODIFIED NUCLEOTIDES ON STRUCTURE OF YEAST TRNA(PHE) - COMPARATIVE-STUDIES BY METAL ION-INDUCED HYDROLYSIS AND NUCLEASE MAPPING, Biochimie, 78(2), 1996, pp. 131-138
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.