Chemical and enzymatic probing of effector-mediated changes in the conformation of a maxizyme

J Inorg Biochem. 2000 Mar;78(4):261-8. doi: 10.1016/s0162-0134(00)00005-2.

Abstract

The protein encoded by chimeric BCR-ABL mRNA causes chronic myelogenous leukemia (CML). We showed previously that a novel allosterically controllable ribozyme, of the type known as a maxizyme, can cleave this mRNA, with high specificity and high-level activity in vivo. We designed the maxizyme in such a way that it was able to form an active core with which to capture the catalytically indispensable Mg2+ ions only in the presence of the BCR-ABL mRNA junction. In order to probe the putative conformational changes, we used a weakly alkaline solution (pH 9.2) in the presence of 25 mM Mg2+ ions to hydrolyze differentially phosphodiester bonds that were located in different environments. Phosphodiester bonds in single-stranded regions were clearly more susceptible to attack by alkali than those within a double-stranded helix. As indicated by earlier data obtained in vivo, our results demonstrated that the active conformation was achieved only in the presence of the junction within the chimeric BCR-ABL mRNA. Moreover, we demonstrated that the use of mild alkaline solutions to probe RNA structures is very informative.

MeSH terms

  • Base Sequence
  • Fusion Proteins, bcr-abl / metabolism
  • Magnesium / metabolism
  • Models, Biological
  • Molecular Sequence Data
  • Nucleic Acid Conformation
  • Protein Conformation
  • RNA, Catalytic / chemical synthesis
  • RNA, Catalytic / chemistry
  • RNA, Catalytic / metabolism*
  • RNA, Messenger / chemical synthesis
  • RNA, Messenger / metabolism*
  • Ribonuclease T1 / pharmacology
  • Temperature
  • Translocation, Genetic

Substances

  • RNA, Catalytic
  • RNA, Messenger
  • Fusion Proteins, bcr-abl
  • Ribonuclease T1
  • Magnesium