(a) The secondary structure of the 56-nt P5abc subdomain. The numbering is the same as in the P4–P6 domain. P5abc has 4 bp deleted from the P4–P6 domain between the C145⋅G158 base pair of P5b and the L5b tetraloop GAAA (nucleotides 150–153); the 4-bp deletion causes the discontinuity in the numbering of this region. The disk between each base pair represents the observed imino proton of the base pair. The terminal G130 imino proton resonance is not observed by NMR because of the fraying of the G130⋅C193 base pair. Each arrow represents an NOE connectivity between two imino protons; the direction and color of the arrows match those of NOE walks in the 2D spectrum shown in b. The dotted arrow between the G134 and U190 imino protons indicates our inability to see the NOE between them caused by the near identity of their chemical shifts. (b) The 1D imino proton spectrum (Upper) and 2D 120-ms NOESY spectrum (Lower) of 2.5 mM of P5abc in 10 mM sodium phosphate and 0.01 mM EDTA (pH 6.4) in 90% H₂O/10% ²H₂O at 10°C. The 1D spectrum was acquired with 4K complex points and processed with a 25°-shifted sine bell squared window function. The 2D data were acquired with 4K points in the D1 dimension and 512 points in the D2 dimension, and were processed with a 40°-shifted sine bell squared function. The arrows connecting diagonal peaks and crosspeaks and the NOE connectivities indicate the spatial arrangement of the imino protons in the secondary structure. The NOE walks are color-coded to the stems in the secondary structure. The 3′ inhomogeneity of the molecule, which is caused by the presence of the N and N+1 species, splits the chemical shifts of imino protons of G131, G191, and U190. Peaks G131* and G191* are from the N+1 impurity molecule, a 57-mer. The peak doubling stops at G134 and does not affect the rest of the molecule.

The secondary structures determined by NMR in solution and by x-ray diffraction in a crystal are compared. Nucleotides that change their base pairing are in red. The solution structure represents the unfolded state, whereas the crystal structure represents the folded state with tertiary interactions. Each solid bar denotes a Watson–Crick base pair, and each hollow bar denotes a non-Watson–Crick base pair. The tertiary interactions seen in the crystal (10) include binding of A183 and A184 to the minor groove of P4 (shown as purple arrows). The green disks represent five magnesium ions identified in the crystal (9, 10) that form direct hydrogen bonds (thick green line) and water-mediated hydrogen bonds (thin green lines) with phosphate oxygens (indicated by p) and guanine bases. The tertiary folding causes significant secondary structure rearrangements, including the loss of three G⋅U base pairs, the loss of a GNRA tetraloop, the formation of a single base bulge, and the addition of two G⋅A base pairs.

Mg²⁺-induced folding monitored by imino proton NMR. Spectra of 0.4 mM of P5abc in 10 mM sodium phosphate and 0.05 mM EDTA (pH 6.4) were measured at 20°C as a function of added MgCl₂. The total concentration of Mg²⁺ was 0 mM (a), 10 mM (b), 20 mM (c), and 30 mM (d). Solid and dashed lines represent the NOE connectivities as determined by 2D NOESY of 3 bp in stem P5b (G141⋅U162, U142⋅A161, and C143⋅G160) for the unfolded and folded conformations, respectively. With the addition of Mg²⁺, the four imino proton peaks of these base pairs decrease, and a new set of four imino proton peaks with the same NOE connectivities appear at different chemical shifts. This response is characteristic of a slow exchange between the folded and unfolded conformations. The percentages of folding estimated from the relative intensity of U142 in the folded and unfolded states are ≈50% at 20 mM Mg²⁺ and 80% at 30 mM Mg²⁺. Six imino proton peaks of three G⋅U pairs (G188⋅U135, G164⋅U177, and G174⋅U167) disappear during the folding; the peaks are indicated by arrows. NOESY spectra of c and d show that these six bases no longer form G⋅U pairs in the folded state. An asterisk indicates the sheared G⋅A base pair of tetraloop L5c that disappears on folding.