Mn²⁺−Nitrogen Interactions in RNA Probed by Electron Spin−Echo Envelope Modulation Spectroscopy:  Application to the Hammerhead Ribozyme

Abstract

We report application of electron spin−echo envelope modulation (ESEEM) spectroscopy to the problem of metal coordination environments in structured RNA molecules. ESEEM has been used in conjunction with 15N-guanosine labeling to identify nitrogen ligation to a Mn2+ site in a hammerhead ribozyme and in Mn2+−model guanosine monophosphate (GMP) complexes. Hammerhead ribozyme complexes consisting of a 34-nucleotide RNA enzyme strand annealed to a 13-nucleotide DNA substrate strand were poised in 1 M NaCl as a 1:1 complex with Mn2+, conditions previously determined to populate a single high-affinity Mn2+ site (Horton, T. E.; Clardy, R. D.; DeRose, V. J. Biochemistry 1998, 51, 18094−18108). Significant modulation of the electron spin−echo from several low-frequency features is detected for the natural-abundance, 14N-hammerhead samples. At 3600 G, the main hammerhead three-pulse ESEEM features arise at 0.6, 1.9, 2.5, and 5.2 MHz and are nearly identical for a Mn2+−GMP complex under the same conditions. For a ribozyme having 15N-guanosine incorporated into the enzyme strand, as well as for an 15N-labeled Mn2+−GMP complex, the modulation is completely altered and consists of one main feature at 3.4 MHz and a smaller feature at the ν n (15N) Larmor frequency of 1.6 MHz. Preliminary analysis of the ESEEM data reveals an apparent hyperfine coupling of A(14N) ∼ 2.3 MHz, similar to previously reported values for Mn2+ directly coordinated to histidine and imidazole. These data demonstrate the potential for ESEEM as a spectroscopic tool for metal ligand determination in structured RNA molecules.

Multifrequency pulsed EPR studies of biologically relevant manganese(II) complexes

“…Recent results from ESEEM experiments performed by our laboratory using site-specifically labeled RNA have precisely identified this binding site as the A9/G10.1 site [99]. The sole nitrogenous RNA-based ligand identified in earlier studies [94,109] is for the first time conclusively attributed to the N7 from G10.1. Another oxygen atom, likely from the phosphate 5′ to the adjacent adenosine (A9) also binds, making the pseudo-octahedral Mn(II) ion coordinatively saturated and rationalizing the relatively small ZFS constants determined from spectral simulation ( Table 1).…”

“…All samples were buffered to pH 7.0 using 10 mM sodium dimethylarsino oxide (sodium cacodylate) and cryoprotected with 20% ethylene glycol before being immediately (<20 s) frozen in liquid nitrogen. The 34-nucleotide RNA enzyme (Dharmacon Research Inc., Boulder, CO) and 13-nucleotide DNA substrate strands (Integrated DNA Technologies) were purified as described previously [92,94,104]. HH hybrids were formed by adding equimolar amounts of enzyme and substrate oligomers and heating to 90 °C for 3 min, and then they cooled slowly to room temperature.…”

“…Certainly some of these metals are needed to aid in the proper folding of the RNA and others are implicated as taking part in the phosphodiester cleavage reaction. Biochemical and EPR spectroscopic studies carried out by DeRose and coworkers [92,94] found that only one of these sites tightly binds its respective metal ion (k d ≤ 10 µM), and that this site must be populated for "catalytic" activity. However, all available spectroscopic evidence indirectly suggested that the high-affinity binding site exists between the N7 of a guanine residue (G10.1) and the phosphate oxygen of an adjacent adenine (A9), about 2.0 nm from the site of RNA cleavage [35,92,[95][96][97][98].…”

Electron-nuclear double resonance spectroscopy (and electron spin-echo envelope modulation spectroscopy) in bioinorganic chemistry

“…Metal ions also are critical to the structure and function of RNA, yet it is quite challenging to define a precise coordination environment in RNA under solution conditions. Thus it is noteworthy that DeRose, Britt, and coworkers (35)(36)(37) recently introduced the use of ENDOR and ESEEM methods to study the catalytic metal ions of ribozymes (RNA-based enzymes).…”

Identification and Characterization of a Divalent Metal Ion-Dependent Cleavage Site in the Hammerhead Ribozyme

“…One example is the incorporation of phosphorothioates (38), which have low affinity for hard metals (e.g., Mg 2+ ) but high affinity for soft metals (e.g., Mn 2+ ), in combination with a functional assay. Spectroscopic methods, such as solution NMR (14,39,40) and EPR (41)(42)(43), have been used to directly detect metal ion binding. Metal ion-induced RNA cleavage has been used to identify metal ion binding sites, such as Pb 2+ (44,45), Tb 3+ (46)(47)(48), and uranyl photocleavage (49,50).…”