The structural basis for mRNA recognition and cleavage by the ribosome-dependent endonuclease RelE

Abstract

Translational control is widely used to adjust gene expression levels. During the stringent response in bacteria, mRNA is degraded on the ribosome by the ribosome-dependent endonuclease, RelE. The molecular basis for recognition of the ribosome and mRNA by RelE and the mechanism of cleavage are unknown. Here, we present crystal structures of E. coli RelE in isolation (2.5 A) and bound to programmed Thermus thermophilus 70S ribosomes before (3.3 A) and after (3.6 A) cleavage. RelE occupies the A site and causes cleavage of mRNA after the second nucleotide of the codon by reorienting and activating the mRNA for 2'-OH-induced hydrolysis. Stacking of A site codon bases with conserved residues in RelE and 16S rRNA explains the requirement for the ribosome in catalysis and the subtle sequence specificity of the reaction. These structures provide detailed insight into the translational regulation on the bacterial ribosome by mRNA cleavage.

Figure 1

Overview of the RelE-Bound 70S Ribosome (A) Top view of the 70S ribosome with the 50S (blue) and 30S (wheat) subunits surrounding RelE (A site, blue), tRNA^fMet (P site, green), a noncognate tRNA^fMet (E site, red), and mRNA (magenta). (A)–(C) are based on the precleavage structure. (B) Close-up of the A and P sites of the 30S subunit viewed from the interface to the 50S. RelE (blue cartoon) spans the 16S rRNA from the head (helix 31 region, green) to the body (helix 18, pink). The mRNA is shown in purple sticks, and the P and E site tRNAs colored as in (A). (C) Close-up view of the A and P sites showing RelE (blue Cα trace), mRNA (purple sticks), and P site tRNA (green cartoon) along with the DF_o-mF_c electron density of the precleavage structure contoured at 1.5 σ. The mRNA sequence is indicated. (D) The postcleavage structure showing the position of the 2′-3′ cyclic phosphate generated upon cleavage (2′-3′ cP). The map is contoured at 1.2 σ. See also Figure S1.

Figure 2

Interactions with rRNA and mRNA (A) Interactions between RelE (blue) and the head region of the 30S (helix 31, wheat). (A)–(E) are based on the precleavage structure. All rRNA references correspond to the E. coli sequences. (B) Interactions between RelE and the body region of the 30S (helix 18, wheat). (C) Interactions between RelE (blue, semitransparent surface with interacting residues shown as sticks) and the decoding center bases A1492/A1493 (green sticks) of 16S rRNA helix 44 (wheat). The unbound conformation of the decoding site is shown with white sticks. (D) Overview of the contacts between RelE and mRNA. RelE is shown as a white cartoon with basic residues near the mRNA as blue sticks. Residues known to affect the activity of P. horikoshii RelE are shown in green. The normal mRNA path is shown with white sticks and the RelE-bound mRNA path with purple sticks. The position of the R81 side chain is inferred from the Cβ position. (E and F) Details of the interactions with mRNA in the precleavage (E) or post-cleavage (F) state showing RelE (blue) with relevant side chains as sticks, P site tRNA (green cartoon), the 16S conserved rRNA helix 34 bulge (wheat), and the mRNA (purple sticks) with the A and P site codons labeled. C1054 is shown in red. The positions of the side chains of R45 and R81 are inferred from Cβ positions and are therefore shown in gray. See also Figure S2.

Figure 3

In Vitro mRNA Cleavage Assay on the 70S Ribosome (A) Sequence of E. coli RelE with the conservation among homologs indicated as increasing strength of red color and the conserved tyrosine at the C terminus in light blue. The secondary structure is shown above the sequence and the interactions to rRNA and mRNA below (all numbers correspond to the E. coli 16S sequence). Residues in the P. horikoshii RelE homolog that affect the activity are indicated with black boxes (Takagi et al., 2005). (B) Overview of the mRNAs used for the in vitro cleavage assays. The 25 nt mRNA consists of a Shine-Dalgarno element (SD) followed by a spacer and the P site (AUG) and A site (UAG) codons. “Trunc Asite” ends after the P site codon with a 3′-OH. The table shows predicted masses of full length mRNA and fragments that would result from cleavage after position 1 or 2 of the A site codon leaving either a 3′-OH, 3′-phosphate (3′-P), or 2′-3′ cyclic phosphate (2′,3′-cP). (C) MALDI mass spectrometry spectra and masses of RNA fragments isolated from complexes in the absence (blue) or presence (red) of RelE. (D) In vitro cleavage assay using 5′ ³²P-labeled mRNA substrates. • is the 25 nt unmodified mRNA; MAP has phosphorothioate linkages after A site codon positions 1 and 2; MAO, MAO2, and MAO3 are 2′-O-methylated at position 1, positions 1 + 2, or all three positions, respectively; and MAD contains a deoxyribose at position 1. The mRNAs were incubated with either T. thermophilus (T.th.) or E. coli (E.co.) 70S ribosomes, tRNA^fMet, and either RelE^wt or RelE^R81A/R45A (RelE^dm) as indicated for either 1 hr (lanes 1–16) or overnight (lanes 17 and 18). The size markers indicate the positions of the full-length (25 nt 3′-OH) and Trunc Asite (18 nt 3′-OH) RNAs and the 20 nt 2′-3′ cyclic phosphate cleavage product, which runs approximately 1 nt faster than the corresponding 3′-OH species. See also Figure S3.

Figure 4

Comparison of the Active Sites of RelE and RNase T1 (A) The active site of RNase T1 (orange cartoon) with critical amino acids shown as labeled sticks. The 3′-phosphate guanosine representing the 3′ end of the cleaved RNA and the guanosine representing the 5′ end are shown as purple sticks. Movements of protons during the reaction are shown with straight arrows (marked H⁺), and the nucleophilic attack of the 2′ oxyanion with a curved arrow. (B) The corresponding region in RelE (blue cartoon) with residues implied in catalysis as blue sticks, and other nearby basic residues as yellow sticks. Residues critical for the activity of P. horikoshii RelE are marked with a ^∗ (Takagi et al., 2005). The nucleotides at positions 2 and 3 of the A site codon are shown as purple sticks and the scissile bond is marked with an arrow. The water molecule near the 2′-OH of position 2 (A20) is shown as a red sphere. See also Figure S4.

Figure 5

A Mechanism for Ribosome-Dependent mRNA Cleavage by RelE (A) Relative inhibition of cleavage in RelE mutants shown as % uncleaved mRNA after 15 min incubation at 37°C, mean value ± SEM. Below, a denaturing RNA gel showing substrate and products for each mutant. (B) Proposed reaction mechanism for the cleavage of mRNA by RelE with residues from RelE shown in blue, 16S C1054 in green, and the mRNA in purple. Stacking of the second A site base with Y87 and the third base with 16S rRNA nucleotide C1054 (double arrows) first orients the RNA correctly for an inline attack. A high local concentration of positive charge shifts the pK_a of Y87 to allow it to act as a general base and abstract a proton from the 2′-OH promoting its attack on the phosphate between A site positions 2 and 3. The negatively charged bipyramidal transition state is stabilized by R61, while R81 acts as a general acid to protonate the 5′ OH leaving group, generating a 2′-3′ cyclic phosphate at the new 3′ end.

Figure 6

Overview of the RelE Cleavage Mechanism RelE (blue) occupies the A site where it pulls the mRNA (purple) into its active site (arrows). Here, the second nucleotide (“2”) stacks with Y87 (blue), while the third nucleotide (“3”) stacks with C1054 (orange). Relevant basic side chains and the water molecule in the active site are shown. The red arrow indicates the nucleophilic attack of the 2′-O⁻.