Binding of manganese(II) to a tertiary stabilized hammerhead ribozyme as studied by electron paramagnetic resonance spectroscopy.
Electron paramagnetic resonance (EPR) spectroscopy is used to study the binding of MnII ions to a tertiary stabilized hammer-head ribozyme (tsHHRz) and to compare it with the binding to the minimal hammerhead ribozyme (mHHRz). Continuous wave EPR measurements show that the tsHHRz possesses a single high-affinity MnII binding site with a KD of < or =10 nM at an NaCl concentration of 0.1 M. This dissociation constant is at least two orders of magnitude smaller than the KD determined previously for the single high-affinity MnII site in the mHHRz. In addition, whereas the high-affinity MnII is displaced from the mHHRz upon binding of the aminoglycoside antibiotic neomycin B, it is not from the tsHHRz. Despite these pronounced differences in binding, a comparison between the electron spin echo envelope modulation and hyperfine sublevel correlation spectra of the minimal and tertiary stabilized HHRz demonstrates that the structure of both binding sites is very similar. This suggests that the MnII is located in both ribozymes between the bases A9 and G10.1 of the sheared G . A tandem base pair, as shown previously and in detail for the mHHRz. Thus, the much stronger MnII binding in the tsHHRz is attributed to the interaction between the two external loops, which locks in the RNA fold, trapping the MnII in the tightly bound conformation, whereas the absence of long-range loop-loop interactions in the mHHRz leads to more dynamical and open conformations, decreasing MnII binding.
Kisseleva
Natalia
N
Khvorova
Anastasia
A
Westhof
Eric
E
Schiemann
Olav
O
D001483:Base Sequence; D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy; D005601:Framycetin / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008345:Manganese / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism*
D016337:RNA, Catalytic; C427204:hammerhead ribozyme; D008345:Manganese; D005601:Framycetin