Cleavage of tRNA with imidazole and spermine imidazole constructs: a new approach for probing RNA structure.
Hydrolysis of RNA in imidazole buffer and by spermine-imidazole conjugates has been investigated. The RNA models were yeast tRNA(Asp) and a transcript derived from the 3'-terminal sequence of tobacco mosaic virus RNA representing a minihelix capable of being enzymatically aminoacylated with histidine. Imidazole buffer and spermine-imidazole conjugates in the presence of free imidazole cleave phosphodiester bonds in the folded RNAs in a specific fashion. Imidazole buffer induces cleavages preferentially in single-stranded regions because nucleotides in these regions have more conformational freedom and can assume more easily the geometry needed for formation of the hydrolysis intermediate state. Spermine-imidazole constructs supplemented with free imidazole cleave tRNA(Asp) within single-stranded regions after pyrimidine residues with a marked preference for pyrimidine-A sequences. Hydrolysis patterns suggest a cleavage mechanism involving an attack by the imidazole residue of the electrostatically bound spermine-imidazole and by free imidazole at the most accessible single-stranded regions of the RNA. Cleavages in a viral RNA fragment recapitulating a tRNA-like domain were found in agreement with the model of this molecule that accounts for its functional properties, thus illustrating the potential of the imidazole-derived reagents as structural probes for solution mapping of RNAs. The cleavage reactions are simple to perform, provide information reflecting the state of the ribose-phosphate backbone of RNA and can be used for mapping single- and double-stranded regions in RNAs.
Vlassov
V V
VV
Institute of Bioorganic Chemistry, Siberian Division of the Russian Academy of Sciences, Novosibirsk.
Zuber
G
G
Felden
B
B
Behr
J P
JP
Giegé
R
R
D001483:Base Sequence; D001665:Binding Sites; D002021:Buffers; D006868:Hydrolysis; D007093:Imidazoles; D015335:Molecular Probes; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009690:Nucleic Acid Conformation; D012331:RNA, Fungal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012349:RNA, Transfer, Asp / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D013096:Spermine; D014027:Tobacco Mosaic Virus / Q000235:genetics / Q000378:metabolism
D002021:Buffers; D007093:Imidazoles; D015335:Molecular Probes; D012331:RNA, Fungal; D012349:RNA, Transfer, Asp; D012367:RNA, Viral; D013096:Spermine; C029899:imidazole