Solution structure of psi32-modified anticodon stem-loop of Escherichia coli tRNAPhe

Abstract

Nucleoside base modifications can alter the structures and dynamics of RNA molecules and are important in tRNAs for maintaining translational fidelity and efficiency. The unmodified anticodon stem-loop from Escherichia coli tRNA(Phe) forms a trinucleotide loop in solution, but Mg2+ and dimethylallyl modification of A37 N6 destabilize the loop-proximal base pairs and increase the mobility of the loop nucleotides. The anticodon arm has three additional modifications, psi32, psi39, and A37 C2-thiomethyl. We have used NMR spectroscopy to investigate the structural and dynamical effects of psi32 on the anticodon stem-loop from E.coli tRNA(Phe). The psi32 modification does not significantly alter the structure of the anticodon stem-loop relative to the unmodified parent molecule. The stem of the RNA molecule includes base pairs psi32-A38 and U33-A37 and the base of psi32 stacks between U33 and A31. The glycosidic bond of psi32 is in the anti configuration and is paired with A38 in a Watson-Crick geometry, unlike residue 32 in most crystal structures of tRNA. The psi32 modification increases the melting temperature of the stem by approximately 3.5 degrees C, although the psi32 and U33 imino resonances are exchange broadened. The results suggest that psi32 functions to preserve the stem integrity in the presence of additional loop modifications or after reorganization of the loop into a translationally functional conformation.

Figure 1

Sequences of (A) the ψ₃₂-modified and (B) fully modified RNA hairpins corresponding to the anticodon arm of E.coli tRNA^Phe. Nucleotide numbering corresponds to the full-length tRNA^Phe molecule. ψ designates pseudouridine and ms²i⁶A designates (2-thiomethyl, N6-dimethylallyl)-adenine. ψ₃₂-A₃₈ base arrangements for (C) the Watson–Crick base pair and (D) the bifurcated hydrogen bond interaction.

Figure 2

Overlay of the UV melting curves of the unmodified (ACSL) and ψ₃₂–modified RNA (ψ₃₂-ACSL) hairpins. Each of the hairpins appears to exhibit two melting transitions—the broad lower temperature transitions presumably corresponding to destacking of the loop nucleotide bases. The apparent melting temperatures of the unmodified and ψ₃₂–modified RNA hairpins are estimated to be 77.0°C and 73.5°C, respectively.

Figure 3

Imino proton spectra of the (A) unmodified and (B) ψ₃₂-modified ACSL^Phe. The imino resonances of ψ₃₂ and U₃₃ have chemical shifts consistent with Watson–Crick base pairs but are exchange broadened in ψ₃₂-ACSL^Phe. This suggests that the pseudouridine has a small destabilizing effect on the loop leading to increased solvent exposure of the imino groups.

Figure 4

H6/8-to-H1′ region of the 2D 400 ms NOESY spectrum. The base-1′ proton sequential walk is traced with intra-residue peaks labeled. The ψ₃₂ H1′ resonance has a chemical shift of 4.56 p.p.m. and is data not shown. The arrows (a) points to the inter-residue sequential NOE between U₃₃ H1′ and G₃₄ H8 and (b) non-sequential NOE between U₃₃ H1′ and A₃₅ H8. The presence of the sequential NOE is not compatible with a U-turn motif for the loop whereas the non-sequential NOE can be produced by non-U-turn loop conformations.

Figure 5

(A) HNN-COSY and (B) multiple-bond ¹⁵N-¹H HSQC spectra showing intra-residue U H5 to N3 and ψ H6 to N1 correlations. The HNN-COSY shows cross-strand H2-N3 crosspeaks for residues 31–39, 38–32, and 37–33 produced by the Watson–Crick base pair configurations. The syn configuration about the ψ₃₂ glycosidic bond and participation of ψ₃₂ N1H in the ψ₃₂-A₃₈ base pair would produce a ψ₃₂ N1-A₃₈ H2 crosspeak (dashed circle). The U₃₃–A₃₇ crosspeak is weak and is indicative of a weak hydrogen bond.

Figure 6

Stereoview of the superposition of (A) the stems and (B) the loops of the 10 converged ψ₃₂-ACSL^Phe structures. Convergence criteria are given in the text. The views are into the major groove. Only sugar and base heavy atoms are shown and the average r.m.s. deviation for the heavy atoms between the ten structures and the average structure is 1.14 Å. The loop and stem regions are locally well defined, but the propeller twist of the A₃₁–U₃₉ base pair is variable among the structures and slightly increases the r.m.s.d. of the full hairpin.

Figure 7

Stereoview of minimized average structure of ψ₃₂-ACSL^Phe (residues G₃₀ to C₄₀). Hydrogen bonding NH and NH₂ protons of base pairs ψ₃₂-A₃₈ and A₃₁–U₃₉ are colored purple and the exocyclic amino nitrogens (A₃₁ and A₃₈) are green. The pro-R(p) phosphoryl oxygens of A₃₁ and ψ₃₂ (yellow) are predicted to form water-mediated hydrogen bonds with ψ₃₂ N1H and slow exchange of the N1 proton. The bridging water molecule is shown in pink. Explicit waters were not used for the structure calculations, but upper distance bounds derived from crystal structures were applied between ψ₃₂ N1 and the phosphoryl oxygens.

Figure 8

Comparison of residues 31–39 of (A) the solution structure of E.coli tRNA^Phe (42) and the crystal structures of (B) fully modified yeast tRNA^Phe (15,16) and (C) fully modified tRNA^Asp (18,21). ψ₃₂ and A₃₈ in (A) form a Watson–Crick base pair. In (B) and (C), nucleotides C₃₂-A₃₈ and ψ₃₂-C₃₈, respectively, form the bifurcated hydrogen bond. The anticodon loop in (A) adopts the tri-loop conformation whereas the anticodon loops in (B) and (C) adopt the U-turn motif. In E.coli tRNA_Cys, ψ₃₂ is in the syn configuration about the glycosidic bond and the loop forms a U-turn motif (17).