RNA structure analysis using T2 ribonuclease: detection of pH and metal ion induced conformational changes in yeast tRNAPhe.
We describe the use of an enzymic probe of RNA structure, T2 ribonuclease, to detect alterations of RNA conformation induced by changes in Mg2+ ion concentration and pH. T2 RNase is shown to possess single-strand specificity similar to S1 nuclease. In contrast to S1 nuclease, T2 RNase does not require divalent cations for activity. We have used this enzyme to investigate the role of Mg2+ ions in the stabilization of RNA conformation. We find that, at neutral pH, drastic reduction of the available divalent metal ions results in a decrease in the ability of T2 RNase to cleave the anticodon loop of tRNAPhe. This change accompanies an increase in the cleavage of the molecule in the T psi C and in the dihydrouracil loops. Similar treatment of Tetrahymena thermophila 5S ribosomal RNA shows that changes in magnesium ion concentration does not have a pronounced effect on the cleavage pattern produced by T2 RNase. T2 RNase activity has a broader pH range than S1 nuclease and can be used to study pH induced conformational shifts in RNA structure. We find that upon lowering the pH from 7.0 to 4.5, nucleotide D16 in the dihydrouracil loop of tRNAPhe becomes highly sensitive to T2 RNase hydrolysis. This change accompanies a decrease in the relative sensitivity of the anticodon loop to the enzyme. The role of metal ion and proton concentrations in maintenance of the functional conformation of tRNAPhe is discussed.
D000818:Animals; D001483:Base Sequence; D002412:Cations; D004722:Endoribonucleases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000494:pharmacology; D009690:Nucleic Acid Conformation; D012313:RNA; D012335:RNA, Ribosomal; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D013379:Substrate Specificity; D013768:Tetrahymena
D002412:Cations; D012335:RNA, Ribosomal; D012346:RNA, Transfer, Amino Acyl; D012313:RNA; D004722:Endoribonucleases; C022543:ribonuclease T(2); D008274:Magnesium