Mutational analysis of the purine riboswitch aptamer domain

Abstract

The purine riboswitch is one of a number of mRNA elements commonly found in the 5'-untranslated region capable of controlling expression in a cis-fashion via its ability to directly bind small-molecule metabolites. Extensive biochemical and structural analysis of the nucleobase-binding domain of the riboswitch, referred to as the aptamer domain, has revealed that the mRNA recognizes its cognate ligand using an intricately folded three-way junction motif that completely encapsulates the ligand. High-affinity binding of the purine nucleobase is facilitated by a distal loop-loop interaction that is conserved between both the adenine and guanine riboswitches. To understand the contribution of conserved nucleotides in both the three-way junction and the loop-loop interaction of this RNA, we performed a detailed mutagenic survey of these elements in the context of an adenine-responsive variant of the xpt-pbuX guanine riboswitch from Bacillus subtilis. The varying ability of these mutants to bind ligand as measured by isothermal titration calorimetry uncovered the conserved nucleotides whose identity is required for purine binding. Crystallographic analysis of the bound form of five mutants and chemical probing of their free state demonstrate that the identity of several universally conserved nucleotides is not essential for formation of the RNA-ligand complex but rather for maintaining a binding-competent form of the free RNA. These data show that conservation patterns in riboswitches arise from a combination of formation of the ligand-bound complex, promoting an open form of the free RNA, and participating in the secondary structural switch with the expression platform.

Figure 1

(a) Tertiary structure of GRA bound to 2,6-diaminopurine (blue), emphasizing the interactions of L2 and L3 and burial of 2,6-diaminopurine in the center of the three-way junction. (b) Secondary structure of the GRA RNA illustrating the interactions of residues in the thre e-way junction and loop-loop elements. Nucleotides that are >95% conserved in an alignment of 100 sequences found in R_fam 7.0 (31) are shown in red and those that are >80% conserved are in orange. Colored boxes correspond to the residue coloring in subsequent structure figures.

Figure 2

Representative data of 2,6-diaminopurine binding the (a) A35U, (b) C61U, and (c) G37A mutants of GRA RNA at 30 °C in a buffer containing 50 mM K⁺-HEPES, pH 7.5, 100 mM KCl and 10 mM MgCl₂. The c-value (where c = K_a•[RNA]) for each of these experiments is 260, 60, and 2, respectively.

Figure 3

Base quadruple interactions between loop 2 (magenta) and 3 (orange) and the effect of mutations on each. The wild type base interactions from the xpt-pbuX guanine riboswitch are illustrated and the effect of mutations of each base are shown as K_D,rel; measurements from a study by Lemay et al. (22) in parenthesis for comparison. (a) Structure of the C61-G37•(A65•U34) quadruple in which C61 and G37 form a standard Watson-Crick pair into whose minor groove face a non-standard trans Watson-Crick/Hoogsteen pair is docked. (b) Structure of the C60-G38•(A66•A33) quadruple which uses a similar architectural scheme in which a trans Watson-Crick/Hoogsteen pair is docked into the minor groove of a Watson-Crick pair.

Figure 4

Base interactions around the ligand binding pocket; blue residues are part of the P1 helix, green are in J2/3, cyan in J1/2 and yellow in J3/1. The wild type base interactions from the xpt-pbuX guanine riboswitch are illustrated and the effect of mutations of each base are shown as K_D,rel; measurements from a study by Lemay et al. (22) in parenthesis for comparison. (a) Minor groove triple at the top of the P1 helix immediately adjacent to the ligand-binding pocket. (b) The second minor groove triple formed between J2/3 and P1. (c) The water-mediated base triple that defines the top of the ligand binding pocket (the triple shown in (a) is the bottom). The red spheres represent well-ordered solvent that mediates the interaction between A73 and the U22-A52 base pair.

Figure 5

Crystal structures of RNAs containing mutations in the P1 helix. (a) 2F_o−F_c simulated annealing omit map contoured at 1 σ of the A21U,U75A mutant in which residues 21, 50 and 75 (shown as sticks) were omitted from the model used for map calculation. (b) 2F_o−F_c simulated annealing omit map contoured at 1 σ of the A21G,U75C mutant in which residues 21, 50 and 75 (shown as sticks) were omitted from the model used for map calculation. (c) Superposition of the wild type and mutants (superposition performed using all backbone atoms of each of the three structures), emphasizing that the positioning of the bases between the three RNAs is nearly identical.

Figure 6

Crystal structures of RNAs containing mutations in the water-mediated base triple of the three way junction. (a) 2F_o−F_c simulated annealing omit map contoured at 1 σ of the U22C/A52G mutant in which residues 22, 47, 52 and 73 (shown as sticks) were omitted from the model used for map calculation. Residues in J1/2 are shown in cyan, those in J2/3 are in green and in J3/1, yellow. (b) 2F_o−F_c simulated annealing omit map contoured at 1 σ of the U22A/A52U mutant in which residues 22, 47, 52 and 73 (shown as sticks) were omitted from the model used for map calculation. (c) Superposition of the wild type and mutants with the superposition performed using all backbone atoms of each of the three structures. While the positioning of the wild type (green) and U22A/A52U mutants show nearly equivalent base positioning, the C22-G52 base pair (orange) is shifted towards A73, placing G52(N2) and A73(N6) in hydrogen bonding distance.

Figure 7

Probing the free state structure of three-way junction mutant GRA RNAs using N-methylisatoic anhydride (NMIA). Mutants are grouped according to their location in the three-way junction and shown in order of decreasing affinity for DAP (left to right). The lane corresponding to the wild type GRA shown at far left appears only once on the full sequencing gel (Supporting Figure 3) but is shown accompanying each set of mutants (GRA*) in order to illustrate the affect of each mutation; thus all three lanes labeled GRA are the identical lane from the gel. RNA was sequenced by inclusion of ddTTP or ddCTP in a reverse transcription reaction containing GRA to assign the nucleotide position of each modification (note that NMIA modification and the sequencing ladder are offset by one residue). Residues that are labeled correspond to the location of mutations and regions of possible alternative structure formation in the free state that prevents ligand binding in some mutants.

Figure 8

Structure of the U47C mutant. (a) 2F_o−F_c simulated annealing omit map contoured at 1 σ of the U47C mutant in which residue 47 was omitted from the model used for map calculation. Note the relatively poor definition of the ribose sugar and the base in the mutant. (b) Superposition of the wild type (green) and mutant (magenta) structures over all backbone atoms. The U47(N3)-U51(O2) and U47(O4)-A52(N6) hydrogen bonds in the wild type structure are shown with arrows and the interatomic distances (in Ångstroms) is denoted as the left value. The right value reflects the interatomic distances of the equivalent atoms in the U47C structure emphasizing that the cytosine packs against J2/3 in the same fashion despite having unfavorable interactions between these two sets of atoms.

Figure 9

Surface representations of left, GRA RNA (“wt”, PDB ID 2B57); middle, the (A21U, U75A) mutant; right, the U47C mutant. The coloration of the surface represents the atomic B-factors for atoms in the RNA (bottom bar), with blue indicating low thermal disorder (B ~ 20–30) and red indicating high disorder in the lattice (B ~ 70–80). The bottom perspective represents a 90° counterclockwise rotation of the upper perspective.