Global flexibility of tertiary structure in RNA: Yeast tRNA ^Phe as a model system

Abstract

The study of RNA structure using x-ray crystallography or NMR has yielded a wealth of detailed structural information; however, such approaches do not generally yield quantitative information regarding long-range f lexibility in solution. To address this issue, we describe a solution-based method that is capable of characterizing the global f lexibilities of nonhelix elements in RNA, provided that such elements are f lanked by helix (e.g., bulges, internal loops, or branches). The ''phased ratio'' method is based on the principle that, for RNA molecules possessing two variably phased bends, the relative birefringence decay times depend on the f lexibility of each bend, not simply the mean bend angles. The method is used to examine the overall f lexibility of the yeast tRNA Phe core (as unmodified transcript). In the presence of magnesium ions, the tRNA core is not significantly more f lexible than an equivalent length of RNA helix. In the absence of divalent ions, the tRNA core gains f lexibility under conditions where its secondary structure is likely to be largely preserved. The phased ratio approach should be broadly applicable to nonhelix elements in both RNA and DNA and to protein-nucleic acid interactions. Yeast tRNAPhe is a paradigm for the study of tertiary structure in RNA: its crystal structure is well-established (1-3), and under comparable ionic conditions, the crystal and solution structures possess nearly identical anticodon-acceptor (mean) interstem angles (1, 4, 5). However, relatively little is known regarding the global flexibility of the tRNA core. Harvey and coworkers (6) have considered the issue of hinge-type flexibility from the computational perspective, noting that largeamplitude bending motions between the acceptor and anticodon domains are not energetically prohibitive. However, a more recent normal-mode analysis of the bending motions for yeast tRNA Phe suggests that the actual range of hinge-type distortions is likely to be small in the absence of external forces (7), a result that is consistent with the small temperature factors for the crystal structure

Conformational energy and structure in canonical and noncanonical forms of tRNA determined by temperature analysis of the rate of s4U8–C13 photocrosslinking

“…Hydrodynamic experiments indicate an overall more extended shape in the tRNA at magnesium concentrations below 100 mM (Eisinger et al 1970;Danchin and Gueron 1970;Römer et al 1970;Willick and Kay 1971;Robison and Zimmerman 1971). Quantitative measurements made by a hydrodynamic method (Friederich and Hagerman 1997;Hagerman 1997) and by gel mobility shift (Friederich et al 1998) show that above 3 mM Mg 2+ the interstem angle between the acceptor stem and anticodon stem is nearly the same as that observed in the crystal structure, but at micromolar Mg 2+ concentrations the interstem angle is z150°. Favre and coworkers (1975) used UVA irradiation (l>324 nm) of various Escherichia coli tRNAs to activate the 4-thiouridine at position 8 (s 4 U8) and showed that it specifically crosslinks to nucleotide C13.…”

“…The terminal decay time decreased to a minimum at 200 mM Mg 2+ , corresponding to an interstem angle of 70°, and increased a little as the Mg 2+ concentration was increased to 10 mM, corresponding to an angle of 80°-90°. Changes in the tRNA interstem angle and flexibility were also measured by the gel electrophoretic mobility of RNA molecules with the yeast tRNA Phe sequence (Friederich et al 1998). Mobility differences confirmed the Mg 2+ dependence of both the tRNA bend angle and the flexibility.…”

“…The flexibility change could not be unambiguously calculated. However, one model predicted a 50-fold decrease in the torsional elastic constant, a measure of the flexibility, although the values in low Mg 2+ concentrations should also be affected by changes in the persistence length of the RNA (Friederich et al 1998).…”

Comparison and functional implications of the 3D architectures of viral tRNA-like structures

“…2E) and A. thaliana tRNA Val (data not shown) produced shapes reminiscent of two helices meeting at an angle, but with a more elongated conformation than that found in a highresolution tRNA Phe crystal structure (Friederich et al 1998;Shi and Moore 2000). However, application of various methods shows variability in the measured helical angles in the tRNA elbow (Friederich et al 1998). For the purposes of this study, we did not concentrate on the precise angle between these helices, but rather on how well the predicted global shape of the TLS RNAs compared with the predicted global shape of the tRNA.…”

“…Multiple reconstructions of both yeast tRNA Phe ( Fig. 2E) and A. thaliana tRNA Val (data not shown) produced shapes reminiscent of two helices meeting at an angle, but with a more elongated conformation than that found in a highresolution tRNA Phe crystal structure (Friederich et al 1998;Shi and Moore 2000). However, application of various methods shows variability in the measured helical angles in the tRNA elbow (Friederich et al 1998).…”