Cleavage of tRNA with imidazole and spermine imidazole constructs: a new approach for probing RNA structure

Abstract

Hydrolysis of RNA in imidazole buffer and by spermineimidazole conjugates has been investigated. The RNA models were yeast tRNAASP 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 tRNAASP 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 singlestranded 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 singleand double-stranded regions in RNAs.

Conformational Changes in the Solution Structure of the Dengue Virus 5′ End in the Presence and Absence of the 3′ Untranslated Region

“…3A), or (iii) 3Ј UTR with a mutated CS (3ЈUTR-CSmut) (Fig. 3B) as described previously (17), disrupting the 5Ј CS-3Ј CS interaction ( Fig. 3A and B).…”

“…All RNase digestions were performed in the presence of 1 g yeast RNA and with nonlimiting MgCl 2 concentrations (3) and were subsequently phenol-chloroform extracted and ethanol precipitated in the presence of glycogen-tRNA. As strong secondary structures can be resistant to enzymatic hydrolysis (13), we also performed chemical cleavage with 0.4 M imidazole overnight at RT in 40 mM NaCl, 1 mM EDTA, and 10 mM MgCl 2 , followed by precipitation with lithium perchlorate-sodium ac-etate and acetone (17). Imidazole cleaves only single-stranded RNA, while double-stranded RNA remains protected.…”

Sequence Specificity in the Interaction of Bluetongue Virus Non-structural Protein 2 (NS2) with Viral RNA

“…For the enzymatic structure probing, the single-stranded specific RNase T1 was used, which cleaves at the 3Ј of unpaired G residues, and RNase U2, which cleaves at the 3Ј of unpaired A or G with a preference for A's. For the chemical probing we used imidazole (27) and lead acetate (28), which are both single strandedspecific. Reactions were analyzed on denaturing gels of various acrylamide concentrations to maximize the information from each probing reaction.…”

cis and trans Requirements for Rolling Circle Replication of a Satellite RNA

“…Here we mapped singleand double-stranded regions of the entire satRPV RNA by probing with structure-sensitive agents under nondenaturing conditions. Figure 1A shows autoradiograms from monomeric satRPV RNA digested partially with RNase T 1 , which cuts single-stranded Gs (15), or imidazole, which cuts unstructured (mostly single-stranded) nucleotides (50). Although some nonspecific cleavages by RNase T 1 occurred under native conditions, they do not affect interpretation of the data.…”