Structure of the Bacillus subtilis 70S ribosome reveals the basis for species-specific stalling

Abstract

Ribosomal stalling is used to regulate gene expression and can occur in a species-specific manner. Stalling during translation of the MifM leader peptide regulates expression of the downstream membrane protein biogenesis factor YidC2 (YqjG) in Bacillus subtilis, but not in Escherichia coli. In the absence of structures of Gram-positive bacterial ribosomes, a molecular basis for species-specific stalling has remained unclear. Here we present the structure of a Gram-positive B. subtilis MifM-stalled 70S ribosome at 3.5-3.9 Å, revealing a network of interactions between MifM and the ribosomal tunnel, which stabilize a non-productive conformation of the PTC that prevents aminoacyl-tRNA accommodation and thereby induces translational arrest. Complementary genetic analyses identify a single amino acid within ribosomal protein L22 that dictates the species specificity of the stalling event. Such insights expand our understanding of how the synergism between the ribosome and the nascent chain is utilized to modulate the translatome in a species-specific manner.

Figure 1. Cryo-EM structure and molecular model of the B. subtilis MifM-stalled ribosome complex.

(a,b) Schematic of the mifM-yidC2 mRNA illustrating the N-terminal transmembrane (TM) segment (black, helix) and C-terminal stalling region (green) of the MifM leader peptide with the stem-loop structure that sequesters the ribosome-binding site (RBS) of the yidC2 gene (blue). In (b) the multisite ribosome stalling (0, +1, +2 and +3) during translation of MifM maintains the unfolded conformation of the mRNA allowing ribosome binding and induction of yidC2 expression. The MifM stalling sequence (residues 69–89) is shown with critical residues boxed in green. Asterisks indicate stop codons. (c) MifM-stalled ribosome complex used for cryo-EM. (d) Transverse section of the cryo-EM structure of the MifM-SRC (30S, yellow; 50S, grey) showing P-tRNA and MifM nascent chain (green) within the ribosomal tunnel and enlargement where ribosomal proteins L4 (cyan) and L22 (orange) are coloured. (e) Electron density (grey mesh) for selected regions of large subunit ribosomal protein and rRNA of the MifM-SRC. (f) Molecular model of the B. subtilis 70S ribosome.

Figure 2. The path of the mRNA through the B. subtilis MifM-SRC.

(a) Location of Shine–Dalgarno(SD)-anti-SD-like helix on 70S ribosome (30S, yellow; 50S, grey, P-tRNA, green). (b) Codon–anticodon interaction between P-tRNA (dark green) and mRNA (pale green). (c) Electron density (grey mesh) and molecular model for the path of the mifM mRNA (green) from the P-site to back of the 30S subunit (d) where it forms an 8 base pair SD-anti-SD-like helix with the 3' end of the 16S rRNA (blue). (e) Schematic for the non-canonical base-pairing observed in the SD-anti-SD-like helix of the MifM-SRC. (f) Comparison of SD-anti-SD-like helix observed in B. subtilis MifM-SRC (blue) with initiation SD-anti-SD helix (grey) and post-initiation SD-anti-SD helix (red) observed previously.

Figure 3. Interactions of the MifM nascent chain with components of the ribosomal tunnel.

(a–f) Contacts between MifM nascent chain (green) and the ribosome in the (a–c) upper and (d–f) lower region of the ribosomal tunnel. Electron density (mesh) is coloured for MifM (green), L4 (cyan), L22 (orange) and rRNA (grey). In (d–f) the cryo-EM map was filtered to 4 Å resolution and the nascent chain modelled as a backbone trace. (g) Schematic for the GFP–MifM–LacZ reporter used to monitor translational arrest, where stalling prevents β-galactosidase production in B. subtilis in vivo. (h–j) β-galactosidase activity from the GFP–MifM–LacZ reporter (as in g) expressed in B. subtilis strains bearing wildtype (WT) or truncated versions of ribosomal proteins (h) L4, (i) L22 or (j) L23. Error bars indicate the s.d. of three independent biological replicates.

Figure 4. Residue M90 in L22 contributes to the species specificity of MifM stalling.

(a–c) Conservation between B. subtilis and E. coli of (a) 23S rRNA nucleotides A751 (A798 in B. subtilis) and A1321 (A1360 in B. subtilis) within helices H35 and H50, respectively, as well as the tunnel lumen region of ribosomal proteins (b) L4 and (c) L22. In (b,c) similar and identical residues are shaded grey and black, respectively. (d) Overview of relative positions of MifM to tunnel lumen residues of L4 and L22. (e,f) β-galactosidase activity from the GFP–MifM–LacZ reporter (as in Fig. 3g) expressed in B. subtilis strains bearing wildtype (WT) L22 compared with (e) Ec-tip mutants where B. subtilis residues 80–98 are substituted with the equivalent E. coli residues (see d) and then additionally reverted to B. subtilis residues by substitutions I85F/M86R or K90M, or (f) L22 mutants where all possible amino-acid substitutions at position M90 of B. subtilis L22 were generated. Error bars indicate the s.d. of three independent biological replicates. (g) Interaction between the side chain of M90 of L22 with the base of G748 and potential hydrogen bonding (dashed line) between the backbone of M90 and the phosphate-oxygen of A751.

Figure 5. MifM stabilizes the uninduced state of the PTC to inhibit A-tRNA accommodation.

(a) A-tRNA accommodation leads to conformational changes in U2584 and U2585 shifting the PTC from an uninduced (yellow) to an induced state (salmon). (b) The PTC of the MifM-SRC (grey density with rRNA (blue) and P-tRNA (green)) resembles that of the uninduced state (yellow). (c) Rotation of U2506 is required to accommodate the shift in U2585 that occurs on A-tRNA accommodation. (d) E87 of MifM nascent chain (green) occupies the position of the induced state of U2506. (e) A-tRNA accommodation leads to a slight shift in the position of A2602. (f) The position of A2602 (blue) in the MifM-SRC encroaches on the A-tRNA binding site and is distinct from the position of A2602 in the uninduced (yellow) or induced states (pink). (g) A distinct conformation of A2602 (yellow) is required to position the GGQ motif of RF2 at the PTC to catalyse peptidyl-tRNA hydrolysis. (h) The position of A2602 (blue) in the MifM-SRC is incompatible with the canonical binding position of RF2 at the PTC.

Figure 6. Molecular basis for the specificity and mechanism of MifM-dependent translation arrest.

Model illustrating how interactions of MifM within the ribosomal tunnel stabilize a specific conformation of E87 that prevents conversion of the uninduced state to the induced state and thereby blocks aminoacyl-tRNA binding at the A-site of the PTC.