Correct folding of a ribozyme induced by nonspecific macromolecules.
The 50-nucleotide hammerhead ribozyme HH-S was tested for self-cleavage. The self-cleavage was very inefficient, and only 13% of HH-S was transformed to its cleavage products. Surprisingly, the percentage of cleavage of HH-S was increased to 30% when 1 microg of tRNA was added to the reaction mixture (6 microL). Other macromolecules such as DNAs and proteins were examined to see if they also augmented cleavage of HH-S, and it was found that most of the macromolecules tested, except nucleotide monomers, did indeed enhance HH-S cleavage. The self-cleaving reaction was almost saturated in 30 min, and only 13% of HH-S was cleaved at 37 degrees C for a 70-min reaction, indicating that 87% of HH-S was in kinetically trapped inactive conformations. Time courses for the reaction of the HH-S self-cleavage were also measured in the presence of tRNA, an oligodeoxyribonucleotide, or BSA. Cleavage of HH-S, which had already reached a plateau of 13% cleaved, increased gradually after the addition of the effector molecules. The first-order rate constant for the self-cleavage reaction in the absence of an effector was comparable to that in the presence of BSA, indicating that the effector molecules do not affect the chemical step of self-cleavage. These results demonstrate that a variety of nonspecific macromolecules can induce conformational change of the hammerhead even in such a low concentration as 0.003% (w/v). This conformational change may occur by macromolecular collisions, or nonspecific weak interactions between HH-S and effectors. Alternatively, a molecular crowding effect may cause the conformational change.
Nashimoto
M
M
Life Science Research Laboratory, Yokohama, Japan. nashimo@nfri.affrc.go.jp
D001483:Base Sequence; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011092:Polyethylene Glycols / Q000737:chemistry; D016337:RNA, Catalytic / Q000737:chemistry*; D012343:RNA, Transfer / Q000378:metabolism
D016337:RNA, Catalytic; D011092:Polyethylene Glycols; D012343:RNA, Transfer