Effects of phosphorothioate and 2-amino groups in hammerhead ribozymes on cleavage rates and Mg2+ binding.
Mg2+ is important for the RNase activity of the hammerhead ribozyme. To investigate the binding properties of Mg2+ to the hammerhead ribozyme, cleavage rates and CD spectra for substrates containing inosine or guanosine at the cleavage site were measured. The 2-amino group of this guanosine interfered with the rate of the cleavage reaction and did not affect the amount of Mg2+ bound to the hammerhead RNA. The kinetics and CD spectra for chemically synthesized oligoribonucleotides with a Sp or Rp phosphorothioate diester bond at the cleavage site indicated that 1 mol of Mg2+ binds to the pro-R oxygen of phosphate. The binding constant for Mg2+ was about 10(4) M-1, which represents outer-sphere complexation. The hammerhead ribozyme catalyzes the cleavage reaction via an in-line pathway. This mechanism has been proved for RNA cleavage by RNase A by using a modified oligonucleotide that has an Sp phosphorothionate bond at the cleavage site. From these results, we present the reaction pathway and a model for Mg2+ binding to the hammerhead ribozyme.
Koizumi
M
M
Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
Ohtsuka
E
E
D000588:Amines / Q000737:chemistry; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D006151:Guanosine / Q000737:chemistry; D007288:Inosine / Q000737:chemistry; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013873:Thionucleotides / Q000737:chemistry*
D000588:Amines; D016337:RNA, Catalytic; D013873:Thionucleotides; D006151:Guanosine; D007288:Inosine; D008274:Magnesium