Requirement of helix P2.2 and nucleotide G1 for positioning the cleavage site and cofactor of the glmS ribozyme

Abstract

The glmS ribozyme is a catalytic RNA that self-cleaves at its 5'-end in the presence of glucosamine 6-phosphate (GlcN6P). We present structures of the glmS ribozyme from Thermoanaerobacter tengcongensis that are bound with the cofactor GlcN6P or the inhibitor glucose 6-phosphate (Glc6P) at 1.7 A and 2.2 A resolution, respectively. The two structures are indistinguishable in the conformations of the small molecules and of the RNA. GlcN6P binding becomes apparent crystallographically when the pH is raised to 8.5, where the ribozyme conformation is identical with that observed previously at pH 5.5. A key structural feature of this ribozyme is a short duplex (P2.2) that is formed between sequences just 3' of the cleavage site and within the core domain, and which introduces a pseudoknot into the active site. Mutagenesis indicates that P2.2 is required for activity in cis-acting and trans-acting forms of the ribozyme. P2.2 formation in a trans-acting ribozyme was exploited to demonstrate that N1 of the guanine at position 1 contributes to GlcN6P binding by interacting with the phosphate of the cofactor. At neutral pH, RNAs with adenine, 2-aminopurine, dimethyladenine or purine substitutions at position 1 cleave faster with glucosamine than with GlcN6P. This altered cofactor preference provides biochemical support for the orientation of the cofactor within the active site. Our results establish two features of the glmS ribozyme that are important for its activity: a sequence within the core domain that selects and positions the cleavage-site sequence, and a nucleobase at position 1 that helps position GlcN6P.

FIGURE 1

Secondary structures of the Bacillus anthracis and Thermoanaerobacter tengcongensis glmS ribozymes. (a) Proposed secondary structure of the glmS ribozyme from B. anthracis modeled after the B. subtilis structure proposed by Winkler, et al. . The site of cleavage is indicated with an open arrow. The position of the P2.2 pairing is shown in red with an alternative-pairing region in the P2 internal loop indicated in gray. Numbering is relative to the cleavage site. The sequence 5' to the cleavage site is not shown; it was 5'-gggaauucAUUGUAAAUUAUAGA-3', where lowercase letters indicate vector-derived sequence. (b) Secondary structure of the B. anthracis glmS ribozyme based on the crystal structure determined by Cochrane et al. . (c) Secondary structure of the T. tengcongensis glmS ribozyme construct used for crystal structure determination.

FIGURE 2

Binding of GlcN6P and Glc6P in high-resolution crystal structures of the T. tengcongensis glmS ribozyme. (a) Simulated annealing omit |F_o|-|F_c| electron density (contoured at 4.0σ) at 1.7 Å resolution for GlcN6P (blue mesh). Ribozyme nucleotides are depicted according to the color scheme in Figure 1(c). Water molecules and metal ions are shown as free floating blue and green spheres, respectively. (b) Superposition of the structure of the T. tengcongensis GlcN6P-bound glmS ribozyme (blue) and Glc6P-bound glmS ribozyme (yellow) at 1.7 Å and 2.2 Å resolution, respectively. GlcN6P and Glc6P are shown in the standard CPK color scheme.

FIGURE 3

Superposition of the GlcN6P-bound structures of the T. tengcongensis (colored according the scheme in Figure 1(c)) and B. anthracis (dark gray) glmS ribozymes. (a) Detailed view of the nucleotides that comprise the GlcN6P-binding pocket. A ∼1 Å shift of the phosphodiester backbone between the two structures is evident between A50-G53 (A42-G45 in the B. anthracis numbering scheme). Deletion of nucleotide U48 in the B. anthracis ribozyme structure is also apparent. The conformation of the scissile phosphate is very similar between the T. tengcongensis GlcN6P-bound structure (τ = 166°, this work) and the four crystallographically independent structures of the GlcN6P-bound B. anthracis ribozyme (mean τ = 167°, standard deviation 3°, 10). (b) Global view of the overall agreement of the structures of the T. tengcongensis and B. anthracis glmS ribozymes. Large structural variation between the two ribozymes is limited to helical elements distant from the active site core.

FIGURE 4

Testing the P2.2 pairing. (a) Cleavage of ANX1 and the P2.2 mutant ribozymes. Products were separated on a denaturing polyacrylamide gel and the positions of the precursor and 3' product are indicated. The 5' product is not visible. (b) P2.2 Mutations in the cis-cleaving ribozyme. Mutated regions in P2.2 are boxed and lowercase designates the bases that are non-wild type. (c) Kinetics of the cis-cleaving ribozymes. Cleavage of ribozymes ANX1 (circles) and P2.2-5'/3' (triangles) show first-order kinetics and cleaved rapidly (inset). Cleavage of the P2.2-5' mutant (squares) and P2.2-3' mutant (diamonds) are significantly slower than ANX1 and the compensatory mutant. Ribozyme cleavage reaction conditions were 2 mM MgCl₂, 0.1 mM GlcN6P at 25°C, pH 7.5 (HEPES).

FIGURE 5

Cleavage of a short oligonucleotide in trans. (a) Reaction time course for the wild-type sequences. Cleavage products of the 5' end-labeled substrate 7-mer RNA were fractionated on PEI plates in 1M LiCl. The product comigrated with a pAp marker generated by nuclease U2 digestion (not shown). (b) The released 5' nucleotide contains a 2',3'-cyclic phosphate group. Treatment with HCl caused the product of the cleavage reaction to migrate slower at pH 5 (1M LiCl with 0.1M Boric acid pH 5). This result is consistent with a 5' product that contains a cyclic phosphate that is hydrolyzed by acid treatment.

FIGURE 6

Testing the P2.2 interaction in the trans acting ribozyme. (a) Sequences of the proposed trans interaction with mutated regions boxed. Lowercase designates mutated bases. (b) Reaction time courses. Products were fractionated by TLC (PEI) and positions of the uncleaved substrate and cleavage product are indicated on the left. (c) Kinetics of trans-cleaving ribozymes. Cleavage of wild-type enzyme with wild-type substrate (circles, k_obs = 11±1 min⁻¹), wild-type enzyme with altered substrate (squares, 0.7±0.5 × 10⁻⁴ min⁻¹), altered enzyme with wild-type substrate (diamonds, 1.2±0.5 × 10⁻⁴ min⁻¹), and altered enzyme with altered substrate (triangles, 1.3±0.1 × 10⁻¹ min⁻¹). Values are the average of three independent determinations. Reactions were at 25 °C in 10 mM MgCl₂, pH 7.5 (HEPES) and 0.1 mM GlcN6P.

FIGURE 7

Comparison of cleavage rates of substrates containing nucleobase analogs at G1 with various cofactors. Cleavage reaction conditions were 10 mM MgCl₂, 25 °C, pH 7.5 (HEPES) and the cofactor. Cofactor concentrations were 0.1 mM GlcN6P (black), 0.1 mM GlcN (red), 10 mM Serinol (green), and 80 mM TRIS (blue). The observed rate constants are the average of two or more independent determinations and were normalized to cofactor concentration. Structures of each of the nucleobases are shown on the left and the cofactors at the bottom.