Abstract
The precursor of an RNA molecule from T4-infected Escherichia coli cells (p2Sp1 RNA) has the capacity to cleave itself at specific positions ((UpA (137-138) and CpA (170-171)) within a possible loop and stem structure. This specific cleavage requires at least a monovalent cation and nonionic detergents. To confirm that the sequence-specific cleavage occurs autolytically, we studied the selective hydrolysis of RNA using a chemically synthesized 13-mer (GUUUCGUACAAAC) having sequences corresponding to G131-C143 of p2Sp1 RNA. The cleavage occurred at two identical sites (UpA and CpA) of a hairpin loop under physiological conditions and was affected by monovalent or divalent metals, nonionic detergent, salt, pH, and temperature. The hairpin loop domain and specific sequences are necessary for the cleavage of RNA 13-mer (GUUUCGUACAAAC).
Highlights
CpA [170-171]) within a possible loop and stem struc- cation and is enhanced bya nonionic detergent
This specific cleavage requires at least a monova-self-cleavage of p2Spl RNA can be decreased by denaturants lent cation and nonionic detergeTnotsc.onfirm that the such as urea
The mechanism of self-cleavage has not been sequence-specific cleavage occurs autolyticwalelys,tudied the selective hydrolysis of RNA using a chemically synthesized 13-mer (GUUUCGUACAAAC) having sequences corresponding to G1s"C'" of p2Spl RNA
Summary
RNA enzymes, or"ribozymes," to target the degradationof spe- pliedBiosystemsmodel381A synthesizer [27]. The best known is the Tetrahymenuanits were prepared from the reaction of 5'-O-dimethoxytrityl-N-propre-rRNA, which mediates the formation of mature RNA by tected and 2'-O-l-(2-chloroethoxy)ethyl-protectednucleosides [27] or self-catalyzed processingof the initial RNA transcrip(t1). CUGAXGA ( X can be any nucleotide) and GAAAC in the ri- acetate at 37 "C for several hours, extracted with an equal volume of bozyme and GUC of the substrate Manoyf these have beenthe water-saturated pheno1:chloroform(l:l), and precipitated with ethanol subject of nucleotide replacement to study their role in the (2-3 volumes) at -20 "C. cleavage t u r e sa r ea l mechanism[13,14,15,16,17,18,19].
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