Probing Adenine Rings and Backbone Linkages Using Base Specific Isotope-Edited Raman Spectroscopy: Application to Group II Intron Ribozyme Domain V

Abstract

Raman difference spectroscopy is used to probe the properties of a 36-nt RNA molecule, “D5”, which lies at the heart of the catalytic apparatus in group II introns. For D5 that has all its adenine residues labeled with 13C and 15N, and utilizing Raman difference spectroscopy, we identify the conformational sensitive -C-O-P-O-C- stretching modes of the unlabeled bonds adjacent to adenine bases, as well as the adenine ring modes themselves. The phosphodiester modes can be assigned to individual adenine residues based on earlier NMR data. The effect of Mg2+ binding was explored by analyzing the Raman difference spectra for [D5 + Mg2+] minus [D5 no Mg2+], for D5 unlabeled, or D5 labeled with 13C/15N-enriched adenine. In both sets of data we assign differential features to G ring modes perturbed by Mg2+ binding at the N7 position. In the A labeled spectra we attribute a Raman differential near 1450 cm−1 and changes of intensity at 1296 cm−1 to Mg binding at the N7 position of adenine bases. The A and G bases involved in Mg2+ binding again can be identified using earlier NMR results. For the unlabeled D5, a change in the C-O-P-O-C stretch profile at 811 cm−1 upon magnesium binding is due to a “tightening up” (in the sense of a more rigid molecule with less dynamic interchange among competing ribose conformers) of the D5 structure. For adenine labeled D5, small changes in the adenine backbone bond signatures in the 810 – 830 cm−1 region suggest small conformational changes occur in the tetraloop and bulge regions upon binding of Mg2+. The PO2− stretching vibration, near 1100 cm−1, from the non-bridging phosphate groups, probes the effect of Mg2+-hydrate inner-sphere interactions that cause an up-shift. In turn, the up-shift is modulated by the presence of monovalent cations since in the presence of Na+ and Li+ the up-shift is (23±2 cm−1) while in the presence of K+ and Cs+ it is (13±3 cm−1), a finding that correlates with the differences in hydration radii. These subtle differences in electrostatic interactions may be related to observed variations in catalytic activity. For a reconstructed ribozyme comprising domains 1–3 (D123) connected in cis plus domain 5 (D5) supplied in trans, cleavage of spliced exon substrates in the presence of magnesium and K+ or Cs+ is more efficient than that in the presence of magnesium with Na+ or Li+.

Time-Resolved Raman and Polyacrylamide Gel Electrophoresis Observations of Nucleotide Incorporation and Misincorporation in RNA within a Bacterial RNA Polymerase Crystal

“…A unique finding from the Raman data is that removing noncovalently bound *UTP by soak-out has marked effects on nucleic acid and protein conformations. The striking new features in Figure 6 (blue, soak-out) at 805 and 783 cm −1 are due to changes in RNA and DNA backbone conformations, 33,44 respectively. They are maximal at 20−25 min and decrease to a plateau after 1 h (Figure 9a,b).…”

“…44 In this context, it is noteworthy that A and B forms of DNA have a relatively intense Raman backbone mode near 785 cm −133,36,47 while the A form of RNA has an intense backbone mode near 810 cm −1 . 44 Using these criteria, it is apparent that *G incorporation is not accompanied by major changes in protein or nucleic acid secondary structure after the experimental dead time (Figure 3, pink). With the exception of the poorly defined feature near 1655 cm −1 , which may be due to an increase in α-helical content following incorporation, there appears to be little change in secondary structure.…”

Raman characterization of Avocado Sunblotchviroid and its response to external perturbations and self-cleavage

“…However, in the presence of low salt and 75% humidity, the symmetric phosphodiester band at ~813 cm -1 appears in the DNA fiber yielding r conf  = 1 [17]. The same conformation was found for ribozyme D5-PL-RNA where r conf  = 1.01 [12]. The difference in the r conf parameter between ASBVd(-) and fiber DNA indicates that although both nucleic acids are in the A conformation, they differ in subtle bond-stretching vibrations located in the –C–O-P-O–C– networks and possibly also in the ribose ring [17,32].…”

Deconvolution of Raman spectroscopic signals for electrostatic, H-bonding, and inner-sphere interactions between ions and dimethyl phosphate in solution

“…Measurement of ν s PO 2 - M and ν s PO 2 - H by peak fitting of the observed Raman spectrum, however, is difficult under conditions where the fraction of coordinated phosphodiester ( f coord ) is small (< 1-2%, Figure 6). Levels of metal coordination, however, can be higher at specific phosphodiesters within a larger structure and can be isolated by site-specific atomic or isotopic substitution[26, 27]. Alternatively, information about the effects of electrostatic or coordination interactions may be obtained from the quantitative analysis of the attenuation of the large ν s PO 2 - signal itself.…”