Chemical probing of tDNAPhe with transition metal complexes: A structural comparison of RNA and DNA

Abstract

The three-dimensional folding of tDNAPhe has been examined and compared to native tRNAPhe using a series of shape-selective transition metal complexes as chemical probes of nucleic acid structure. Rh(phen)2 phi 3+ (phen = phenanthroline, phi = 9,10-phenanthrenequinonediimine), which targets sites of tertiary interaction in tRNAPhe, cleaves specifically at similar sites on tDNAPhe. However, this rhodium complex also targets 5'-pyr-pyr-pur-3' sites within the acceptor and anticodon stems of tDNA;Rh(phen)2 phi 3+ generally targets 5'-pyr-pyr-pur-3' sites in B-form duplex DNA. On tRNAPhe, Rh(DIP)3(3+) (DIP = 4,7-diphenyl-1,10-phenanthroline) specifically cleaves C70, which neighbors a GU mismatch, and targets psi 55 as well, within the hydrophobic region of tRNAPhe. On tDNAPhe no specific cleavage by Rh(DIP)3(3+) is observed. The cleavage studies, taken together, indicate that globally the tertiary folding of tDNAPhe resembles that of tRNAPhe. However, the double helical regions of the DNA analog differ from tRNAPhe, likely in adopting a more B-like conformation. As a consequence, the GT mismatch within the acceptor stem of tDNA does not present the same recognition elements as in tRNA, and the GT mismatch is no longer recognized by the shape-selective rhodium complex. The present work underscores the utility of applying DNA analogs to studies of RNA structure and function, since the general folding characteristics of the two polymers are likely to be similar. However, structural probing with transition metal complexes offers a valuable companion to such experiments, since the shape-selective probes, with sensitivity, may be used to delineate locally on the polymer those regions which may differ in structure.

The Escherichia coli tRNA-Guanine Transglycosylase Can Recognize and Modify DNA

“…One role of the 2Ј-hydroxyls (in tRNA at least) is to increase the stability of the molecule (12). Studies using transition metal complexes have confirmed that the global structure of tDNA Phe resembles that of tRNA Phe but that the base paired stem conformations differ somewhat (13 (14). All of the above studies were conducted using canonical base replacements (A, C, G, T, and deoxyribose for the tDNA analogues) and demonstrate that tDNA and tRNA can have similar conformations in solution.…”

An mRNA Loop/Bulge in the Ferritin Iron-responsive Element Forms in Vivo and Was Detected by Radical Probing with Cu-1,10-phenantholine and Iron Regulatory Protein Footprinting

“…If the metal is redox active at physiological conditions, as it is for copper, radical cleavage should occur at the MC/RNA binding site to report on the RNA shape. In solution, Cu-phen binds at RNA loops, bulges, and helix distortions in tRNA, mRNA, and rRNA that are indistinguishable for most alkylating agents and too small for access by protein nucleases (3)(4)(5)(6)(7). We now show that Cu-phen, an MC, detects the tertiary structure in ferritin mRNA in HeLa cells selectively distinguishing the wild type IL/B from a mutant IRE.…”

“…Do mRNA tertiary structures such as the ferritin mRNA IRE form in vivo? Metal coordination complexes (MCs), protein nucleases, and alkylating agents have all been used to analyze mRNA solution structure, but MCs are particularly sensitive to the tertiary structure or shape of the RNA binding site (3)(4)(5)(6)(7). MCs have specific geometry and relatively rigid shapes contributed by small organic molecules, coordinated to a metal ion (Fig.…”

The Thermodynamics of DNA Structural Motifs

“…For DNA, however, the database of secondary structures determined by physical means or by comparative sequence analysis is much smaller (7,10,11,14,19,27,30,31,35,43,61,78). Table 5 shows the secondary structure prediction results for the currently available DNA database.…”