Common and distinctive features of GNRA tetraloops based on a GUAA tetraloop structure at 1.4 A resolution

Abstract

GNRA tetraloops (N is A, C, G, or U; R is A or G) are basic building blocks of RNA structure that often interact with proteins or other RNA structural elements. Understanding sequence-dependent structural variation among different GNRA tetraloops is an important step toward elucidating the molecular basis of specific GNRA tetraloop recognition by proteins and RNAs. Details of the geometry and hydration of this motif have been based on high-resolution crystallographic structures of the GRRA subset of tetraloops; less is known about the GYRA subset (Y is C or U). We report here the structure of a GUAA tetraloop determined to 1.4 A resolution to better define these details and any distinctive features of GYRA tetraloops. The tetraloop is part of a 27-nt structure that mimics the universal sarcin/ricin loop from Escherichia coli 23S ribosomal RNA in which a GUAA tetraloop replaces the conserved GAGA tetraloop. The adenosines of the GUAA tetraloop form an intermolecular contact that is a commonplace RNA tertiary interaction called an A-minor motif. This is the first structure to reveal in great detail the geometry and hydration of a GUAA tetraloop and an A-minor motif. Comparison of tetraloop structures shows a common backbone geometry for each of the eight possible tetraloop sequences and suggests a common hydration. After backbone atom superposition, equivalent bases from different tetraloops unexpectedly depart from coplanarity by as much as 48 degrees. This variation displaces the functional groups of tetraloops implicated in protein and RNA binding, providing a recognition feature.

FIGURE 1.

Structural overview and lattice contacts. (A) Schematic of the RNA sequence in the crystal structure showing the tetraloop (green), bulged-G motif (yellow), the flexible region (blue), and the stem region (red). Gray arrows indicate the two adenosines and WC base pairs involved in lattice contacts that mimic A-minor like interactions (Fig. 3 ▶). The sites of mutation are underlined. (B) A ribbon drawing of the GUAA mutant structure.

FIGURE 2.

Comparison of the GUAA structure (thick lines) with other GNRA tetraloop structures. (A) Stereodiagram of the backbone atom superposition of 20 tetraloop structures determined to at least 2.4 Å resolution (see Materials and Methods); the standard orientation of the tetraloop bases (purple) is contrasted with the altered orientation (green). (B) Two extremes in the conformation of the base pair between the first and the fourth nucleotide of the tetraloop. (C) Stereodiagram of the superposition of the GUAA tetraloop reported here (green) and the other tetraloop structures determined to at least 2.0 Å resolution (see Materials and Methods). The GUAA structure illustrates the altered orientation of the last three bases of the tetraloop whereas the other structures illustrate the standard orientation. In the tetraloop, the pro-R_p oxygen of the third nucleotide (circled) is within 3.2 Å of the first guanine base. Arrows marking solvent-mediated hydrogen bonds are pointing toward proton acceptors. Most of the conserved solvent sites are obligate proton acceptors (asterisks).

FIGURE 3.

An interaction that mimics an A-minor motif. (A) Stereoview of the GUAA tetraloop (green) docking into the minor groove of a symmetry related stem region (red). The asterisk marks a solvent site that is conserved in tetraloop structures. The refined models of type I (B) and type II (C) A-minor contacts are superimposed on a 1.4 Å SIGMAA-weighted 2F_o–F_c electron density map. Weak C-H…O hydrogen bonds (as reviewed in Desiraju 1996) between C2 of A2662 and N3 of G2669 (3.2 Å) and between C2 of A2661 and O2 of C2652 (3.2 Å) may contribute to the stability of this A-minor interaction.