Chemical and enzymatic probing of effector-mediated changes in the conformation of a maxizyme.
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.
Zhou
J M
JM
Institute of Applied Biochemistry, University of Tsukuba, Tsukuba Science City, Japan.
Nakamatsu
Y
Y
Kuwabara
T
T
Warashina
M
M
Tanaka
Y
Y
Yoshinari
K
K
Taira
K
K
D001483:Base Sequence; D016044:Fusion Proteins, bcr-abl / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D016337:RNA, Catalytic / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D012333:RNA, Messenger / Q000138:chemical synthesis / Q000378:metabolism*; D006163:Ribonuclease T1 / Q000494:pharmacology; D013696:Temperature; D014178:Translocation, Genetic
D016337:RNA, Catalytic; D012333:RNA, Messenger; D016044:Fusion Proteins, bcr-abl; D006163:Ribonuclease T1; D008274:Magnesium