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 transcript and the U8G mutant have been investigated using metal ion-induced hydrolysis and nuclease digestion. Differences in the solution structure 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 molecule involve 50-fold enhancement of S1 nuclease digestion at p36, weak cleavages in the D-loop and lack of some digestion sites in the T-loop. The nuclease V1 digestion patterns are very similar for studied molecules. Changes in the pattern of hydrolysis of the D-loop caused by mutation of the conservative base U8 to G are detected by metal-induced hydrolysis only. Our results indicate clearly that metal ions and enzymatic probes monitor different features of RNA structure and their combined use is highly advantageous in studying subtle structural changes in tRNA.
Michałowski
D
D
Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland.
Wrzesinski
J
J
Ciesiołka
J
J
Krzyzosiak
W J
WJ
D001483:Base Sequence; D004722:Endoribonucleases / Q000378:metabolism*; D005063:Europium / Q000494:pharmacology; D006868:Hydrolysis; D007854:Lead / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008670:Metals / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation / Q000187:drug effects*; D012346:RNA, Transfer, Amino Acyl / Q000737:chemistry*; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism*
D008670:Metals; D012346:RNA, Transfer, Amino Acyl; D007854:Lead; D008345:Manganese; D005063:Europium; D004722:Endoribonucleases; C032825:ribonuclease V(1); D015719:Single-Strand Specific DNA and RNA Endonucleases; D008274:Magnesium