Anionic G•U pairs in bacterial ribosomal rRNAs

Abstract

Wobble GU pairs (or G•U) occur frequently within double-stranded RNA helices interspersed between standard G=C and A-U Watson-Crick pairs. Another type of G•U pair interacting via their Watson-Crick edges has been observed in the A site of ribosome structures between a modified U34 in the tRNA anticodon triplet and G + 3 in the mRNA. In such pairs, the electronic structure of the U is changed with a negative charge on N3(U), resulting in two H-bonds between N1(G)…O4(U) and N2(G)…N3(U). Here, we report that such pairs occur in other highly conserved positions in ribosomal RNAs of bacteria in the absence of U modification. An anionic cis Watson-Crick G•G pair is also observed and well conserved in the small subunit. These pairs are observed in tightly folded regions.

Keywords: G•U pair; anionic; anticodon; codon; mRNA; rRNA; tRNA.

FIGURE 1.

(A) A standard wobble G•U pair (from PDB 1HQ1, Batey et al. 2001) with a water molecule in the minor groove (red sphere) and a hydrated potassium ion (purple sphere) in the major groove. All distances are in Å and between the heavy atoms. The distance between the two C1′ carbon atoms is 10.7 Å (values between 10.4 Å and 10.7 Å are commonly observed). (B) The observed G•U pair between G3 and modified U34* in the ternary complex between the ribosomal A site, the anticodon loop of the A-tRNA and the mRNA (from PDB 5E81, Rozov et al. 2016a). The tRNA U34* is modified in 5-methylaminomethyl-2-thioUridine. The distance between the two C1′ carbon atoms is 11.4 Å. (C) Possible electronic structure of the modified U34* where X at position 2 is S and R at position 5 is methylaminomethyl, probably charged and thus forming a zwitterion. The negative charge may be delocalized between the N3 and S2. For chemical data and a discussion, see Sochacka et al. (2015, 2017).

FIGURE 2.

(A) The internal loop of helix h24 in 16S rRNA in contact with the anionic U677•G713 pair is shown in cyan. Please note the stacking of the two G776 and G775 and the usual wobble pair at G778•U804. (B) The anionic U677•G713 pair with the contact to the O2′ of A777 in the 16S rRNA of E. coli (from 8B0X, Fromm et al. 2023). For comparison, the distances in PDB 7K00 (Watson et al. 2020) corresponding to N1(G)…O4(U), N2(G)…N3(U) are 2.7 Å and 3.0 Å and those for N2(G)…O2′(A777), O2(U)…O2′(A777) are 3.1 Å and 2.4 Å. The red spheres represent water molecules in close proximity.

FIGURE 3.

(A) The anionic G664•G741 pair with some water molecules and a hydrated K⁺ ion in the deep major groove. (B) The highly conserved organization around the anionic G664•G741 in E. coli (PDB 8B0X, Fromm et al. 2023). The residues G666•U740 form a usual wobble G•U pair. A665/G724 form a cis Hoogsteen/Watson–Crick pair and G722•G733 a trans Watson–Crick/Watson–Crick pair with G722 in the unusual syn conformation. For additional contacts to G664, see Supplemental Figure S2A.

FIGURE 4.

(A) Part of the secondary structure of the 16S rRNA showing helices h22, h23, h23_1. The blue triangles indicate that those two residues bulge out so that A665 and G724 can form a cis Hoogsteen/Watson–Crick pair. The right-angle arrows indicate the coaxial stacking between the helices in the three-way junction. (B) The figure shows how the shallow minor groove side of the anionic G664•G741 binds tightly to the hairpin h23_1. Drawing based on E. coli (PDB 8B0X, Fromm et al. 2023).

FIGURE 5.

(A) The fold of the three-way junction with the anionic G1099•U1086 pair with, in purple, the contacts between G1108 and the 5′-phosphate of U1095 forcing G1094 to bulge out. (B) Elements of the secondary structure of the three-way junction. The right-angle arrows indicate the coaxial stacking between the helices in the three-way junction. (C) The anionic G1099•U1086 pair with a hydrated Mg²⁺ ion (green sphere) with its hydration sphere (red spheres) and a direct contact with an anionic phosphate oxygen of U1085. The distances to the Mg²⁺ ion are between 1.9 Å and 2.1 Å, except the one to the O6(G1099). Drawings based on E. coli (PDB 8B0X, Fromm et al. 2023).

FIGURE 6.

(A) The anionic G2304•U2312 pair in the 23S rRNA and its contacts with two conserved residues of protein uL5. (B) The drawing illustrates how the capping loop following the anionic pair (in purple) is buried within uL5. Drawings based on E. coli (PDB 8B0X, Fromm et al. 2023). Please note that in 8B0X that pair is numbered G2308•U2316 (see Table 1).

FIGURE 7.

Views of the cryo-EM maps around some anionic pairs based on the E. coli ribosome structure (7K00, Watson et al. 2020). (A) Map around the anionic G2304•U2312 pair in the 23S rRNA and its contacts with two conserved residues of protein uL5, N37, and D153. The nature of the amino acid side chain atoms in contact with G2304 and U2312 cannot be ascertained at this stage. The distance between the nitrogen atom of N37 and one of the oxygen atoms of D153 is 3.4 Å. Note also that the density for the carboxylate group is weak, which could be due to radiation damage (Marques et al. 2019). (B) Map around the anionic U677•G713 pair and A777 in the 16S rRNA. (C) Map around the anionic G664•G741 in E. coli. The water molecules bound to O4(G741) are above and below the plane of the base.

FIGURE 8.

Two views of the molecular environment around the anionic pairs and their proximity within the ribosome structure. The guanines are colored red and shown as van der Waals spheres with the uracils colored in cyan. (A) The A-tRNA and P-tRNA bound to the mRNA (with van der Waals spheres at the right) are shown. The protein uL5 in the 23S rRNA is colored green. (B) The backbone of the whole 16S rRNA is superimposed (with the same color code). The drawings are from 7K00 (Watson et al. 2020).