- Email: [email protected]
Peptides in the Ribosomal Tunnel Talk Back
Molecular Cell
Previews Peptides in the Ribosomal Tunnel Talk Back Daniel N. Wilson1,* 1Gene Center and Department for Biochemistry, Center for Protein Science-Munich (CiPS-M), University of Munich, Feodor-Lynen-Strasse 25, D-81377 Munich, Germany *Correspondence: [email protected] DOI 10.1016/j.molcel.2011.01.017
In this issue of Molecular Cell, Ramu et al. demonstrate that nascent peptides located within the ribosomal tunnel can talk back to the peptidyl transferase center to induce translational stalling by restricting the species of aminoacyl-tRNAs that can bind there. The passage of specific nascent polypeptide chains through the ribosomal tunnel can lead to translational stalling, an event utilized by the cell to regulate gene expression (reviewed by Ito et al., 2010). In some cases, the stalling event requires the presence of an additional effector molecule, such as an antibiotic (Ramu et al., 2009). One well-characterized example is found in the bacterial ermC operon, where subinhibitory concentrations of the macrolide drug erythromycin promote stalling during translation of the upstream leader peptide gene, ermCL (Ramu et al., 2009). The translation arrest permits an alternative mRNA secondary structure to form, which in turn allows initiating ribosomes to translate the downstream ermC gene (Figure 1A). ErmC is a methyltransferase that confers resistance to erythromycin via modification of the ribosomal RNA (rRNA) that comprises the drug-binding site. In this manner, the cell can detect and respond to the threat of an antimicrobial agent by production of the appropriate resistance protein. Despite our general understanding of such translational stalling systems, the mechanism by which the nascent peptide and ligand interact with the tunnel to inactivate the peptidyl transferase center (PTC) remains unclear. In the article by Mankin and coworkers (Ramu et al., 2011), a single amino acid position of the Erm peptides is shown to modulate the ability of the PTC to accommodate specific amino acids at the A-site. Previous studies have demonstrated that in the presence of erythromycin, translation of ermCL stalls at the ninth codon (Ile9), with the peptidyl-tRNAIle located in the P-site (Ramu et al., 2009; Vazquez-Laslop et al., 2008) (Figure 1B). Mutations that abrogate stalling are
located in the 6IFVI9 sequence of ErmCL, whereas stalling occurs independently of the identity of the tenth amino acid at the A-site (Ramu et al., 2009; Vazquez-Laslop et al., 2008). Additionally, alteration of 23S rRNA nucleotides A2062 or A2503 of the ribosomal tunnel abolishes stalling, as does the absence of the cladinose sugar of erythromycin (Va´zquez-Laslop et al., 2008, 2010). Collectively, these data suggest a complex network of interactions between the ErmCL peptide, the cladinose sugar of erythromycin, and components of the ribosomal tunnel, which relays a signal back to the PTC to prevent peptide bond formation between the amino acids Ile9 (P-site) and Ser10 (A-site) (Figure 1B). How the signal is transferred to the PTC and the mechanism of PTC inactivation are addressed by Ramu et al. (2011) by comparing ErmCL stalling with another stalling peptide present in the ermA operon. The ermA gene also encodes a methyltransferase that confers resistance to erythromycin, but unlike the ermC operon, two leader peptides, termed ermAL1 and ermAL2, are located upstream of the ermA gene (Ramu et al., 2009). Mankin and coworkers show that while the ErmAL2 stalling sequence operates analogous to ErmCL, the sequence (Figure 1A) and stalling mechanism of the ErmAL1 peptide differs: Translation of ermAL1 stalls when the eighth codon (Val8) is located in the P-site; however, unlike ErmCL stalling, the identity of the A-site (ninth) amino acid dramatically affects the efficiency of stalling (Ramu et al., 2011). ErmAL1 stalling is most efficient when the ninth amino acid is charged, such as Asp, Lys, Arg, His, or the native Glu, but also occurs with uncharged amino acids Trp, Tyr, and Ile
(Figure 1C). In contrast, when Phe, Met, or Cys is at the ninth position of the ErmAL1 peptide, translational stalling is reduced or abolished (Ramu et al., 2011). Generating chimeric ErmCL-AL1 peptides revealed that the 2 position (Phe7 in ErmCL or Ala6 in ErmAL1) regulates whether the PTC is selective, by allowing incorporation of a subset of amino acids (as in ErmAL1) (Figure 1C), or restrictive, i.e., by preventing incorporation of all amino acids (as in ErmCL [Figure 1D]). The proximity of the 2 position of the Erm peptides to A2062, A2503, and the cladinose of erythromycin led Mankin and coworkers to propose two relays for inactivation of the A-site of the PTC, one operating through U2504 and C2452 and the other through G2061 and A2451 (Ramu et al., 2011) (Figure 1E). A2451 and C2452 form a cleft into which the aminoacyl moiety of the A-tRNA is positioned for peptide bond formation (Figure 1E); therefore, it is easy to envisage how slight alterations in the position of these nucleotides could generate a narrower restrictive or wider selective A-site cleft. With these new insights into the mechanism of action of the Erm peptides in hand, one important future direction will be to assess how generally applicable the results are to other nascent peptide-mediated stalling systems, including those that are drug independent. An obvious candidate is the SecM leader peptide, where translational arrest is also abolished by mutation of the 2 position (Arg163) of the SecM peptide (Yap and Bernstein, 2009) as well as by mutations of A2062 or A2503 of the 23S rRNA (Va´zquez-Laslop et al., 2010). Moreover, a putative contact between A2062 and Arg163 of SecM is observed in the cryoelectron microscopy
Molecular Cell 41, February 4, 2011 ª2011 Elsevier Inc. 247
Molecular Cell
Previews
Laslop et al., 2010), indicating that the relay mechanism of TnaC is likely to be distinct from ErmCL and SecM. Indeed, stalling of other Erm peptides, such as ErmBL and ErmDL, is also independent of A2062/A2503 (Va´zquez-Laslop et al., 2010). In addition, many stalling sequences are longer than the Erm peptides, with critical residues ( 9 to 12 positions) being located deeper in the tunnel (Bhushan et al., 2010, 2011; Cruz-Vera et al., 2005; Seidelt et al., 2009; Yap and Bernstein, 2009). This deeper region of the tunnel is constricted and represents a hot spot for mutations that alleviate translational arrest (Ito et al., 2010). Thus, from the relatively small number of stalling peptides that have so far been identified and characterized, it appears likely that a diverse array of allosteric mechanisms are employed by distinct stalling peptides to attain the common goal: namely, translational arrest. REFERENCES Bhushan, S., Meyer, H., Starosta, A.L., Becker, T., Mielke, T., Berninghausen, O., Sattler, M., Wilson, D.N., and Beckmann, R. (2010). Mol. Cell 40, 138–146.
Figure 1. Mechanistic Insights into ErmCL- and ErmAL-Mediated Translational Arrest (A) In the absence of erythromycin, stem combinations 1 + 2 and 3 + 4 are favored, leading to repression of ermC due to the sequestering of the ribosome-binding site (RBS) and AUG start codon of ermC within the 3 + 4 stem loop. In the presence of subinhibitory concentrations of erythromycin, stalling of the ribosome at the ninth codon (Ile9) during translation of ermCL leads to an alternative 2 + 3 stem loop, allowing ribosomes to access the RBS and AUG of the ermC gene. In contrast, translational stalling of ErmAL1 occurs when the eighth codon (Val8) is in the P-site. (B) Schematic of the ErmCL stalling ribosome, with ErmCL-tRNAIle in the P-site (gold) and the tenth codon encoding Ser-tRNASer located in the A-site. Interaction between erythromycin (blue), the ErmCL peptide, and the ribosome relays a signal (arrowed) back to inactivate the peptidyl transferase center (PTC). (C) In the presence of erythromycin (Ery) and ErmAL1 (Ala is 2 position), the A-site of the peptidyl transferase center (PTC) becomes selective: Peptide bond formation with certain amino acids is abolished (red) or becomes very slow (orange), leading to stalling or partial stalling, but other amino acids are permitted (green), and no stalling ensues. (D) In contrast, ErmCL (Phe at 2 position) renders the A-site restrictive, preventing all amino acids from forming peptide bonds, leading to translational stalling. (E) Two possible signal-relay pathways (arrowed) communicating the information from the ribosomal exit tunnel to the PTC A-site. Erythromycin, blue; ErmAL-tRNA in the P-site (yellow) has the 2 position colored red. The aminoacyl moiety of the A-site tRNA (gray) inserts into the cleft between A2451 and A2452 (light blue) of the 23S rRNA. Other potential nucleotides involved in the relays include A2503 and U2504 (cyan) as well as A2062 and G2061 (teal).
structure of a SecM-stalled ribosome (Bhushan et al., 2011). Curiously, in this structure the CCA end of the SecM-tRNA is mispositioned at the P-site of the PTC (Bhushan et al., 2011), raising the question of whether a similar shift in the Erm-tRNAs also contributes to Erm peptide-mediated arrest. While A-site selectivity has also been observed with the TnaC stalling
peptide (Cruz-Vera et al., 2005), sequence conservation as well as biochemical (CruzVera and Yanofsky, 2008) and structural data (Seidelt et al., 2009) would suggest it is the 3 position (Asp21), rather than 2 position, that is important for arrest. However, it should be noted that mutation of A2062 or A2503 does not abolish TnaCmediated translational stalling (Va´zquez-
248 Molecular Cell 41, February 4, 2011 ª2011 Elsevier Inc.
Bhushan, S., Hoffmann, T., Seidelt, B., Frauenfeld, J., Mielke, T., Berninghausen, O., Wilson, D.N., and Beckmann, R. (2011). PLoS Biol., in press. Published online January 18, 2011. 10.1371/journal. pbio.1000581. Cruz-Vera, L.R., and Yanofsky, C. (2008). J. Bacteriol. 190, 4791–4797. Cruz-Vera, L.R., Rajagopal, S., Squires, C., and Yanofsky, C. (2005). Mol. Cell 19, 333–343. Ito, K., Chiba, S., and Pogliano, K. (2010). Biochem. Biophys. Res. Commun. 393, 1–5. Ramu, H., Mankin, A., and Vazquez-Laslop, N. (2009). Mol. Microbiol. 71, 811–824. Ramu, H., Vazquez-Laslop, N., Klepacki, D., Dai, Q., Piccirilli, J., Micura, R., and Mankin, A. (2011). Mol. Cell 41, this issue, 321–330. Seidelt, B., Innis, C.A., Wilson, D.N., Gartmann, M., Armache, J.P., Villa, E., Trabuco, L.G., Becker, T., Mielke, T., Schulten, K., et al. (2009). Science 326, 1412–1415. Va´zquez-Laslop, N., Thum, C., and Mankin, A.S. (2008). Mol. Cell 30, 190–202. Va´zquez-Laslop, N., Ramu, H., Klepacki, D., Kannan, K., and Mankin, A.S. (2010). EMBO J. 29, 3108–3117. Yap, M.N., and Bernstein, H.D. (2009). Mol. Cell 34, 201–211.
Previews Peptides in the Ribosomal Tunnel Talk Back Daniel N. Wilson1,* 1Gene Center and Department for Biochemistry, Center for Protein Science-Munich (CiPS-M), University of Munich, Feodor-Lynen-Strasse 25, D-81377 Munich, Germany *Correspondence: [email protected] DOI 10.1016/j.molcel.2011.01.017
In this issue of Molecular Cell, Ramu et al. demonstrate that nascent peptides located within the ribosomal tunnel can talk back to the peptidyl transferase center to induce translational stalling by restricting the species of aminoacyl-tRNAs that can bind there. The passage of specific nascent polypeptide chains through the ribosomal tunnel can lead to translational stalling, an event utilized by the cell to regulate gene expression (reviewed by Ito et al., 2010). In some cases, the stalling event requires the presence of an additional effector molecule, such as an antibiotic (Ramu et al., 2009). One well-characterized example is found in the bacterial ermC operon, where subinhibitory concentrations of the macrolide drug erythromycin promote stalling during translation of the upstream leader peptide gene, ermCL (Ramu et al., 2009). The translation arrest permits an alternative mRNA secondary structure to form, which in turn allows initiating ribosomes to translate the downstream ermC gene (Figure 1A). ErmC is a methyltransferase that confers resistance to erythromycin via modification of the ribosomal RNA (rRNA) that comprises the drug-binding site. In this manner, the cell can detect and respond to the threat of an antimicrobial agent by production of the appropriate resistance protein. Despite our general understanding of such translational stalling systems, the mechanism by which the nascent peptide and ligand interact with the tunnel to inactivate the peptidyl transferase center (PTC) remains unclear. In the article by Mankin and coworkers (Ramu et al., 2011), a single amino acid position of the Erm peptides is shown to modulate the ability of the PTC to accommodate specific amino acids at the A-site. Previous studies have demonstrated that in the presence of erythromycin, translation of ermCL stalls at the ninth codon (Ile9), with the peptidyl-tRNAIle located in the P-site (Ramu et al., 2009; Vazquez-Laslop et al., 2008) (Figure 1B). Mutations that abrogate stalling are
located in the 6IFVI9 sequence of ErmCL, whereas stalling occurs independently of the identity of the tenth amino acid at the A-site (Ramu et al., 2009; Vazquez-Laslop et al., 2008). Additionally, alteration of 23S rRNA nucleotides A2062 or A2503 of the ribosomal tunnel abolishes stalling, as does the absence of the cladinose sugar of erythromycin (Va´zquez-Laslop et al., 2008, 2010). Collectively, these data suggest a complex network of interactions between the ErmCL peptide, the cladinose sugar of erythromycin, and components of the ribosomal tunnel, which relays a signal back to the PTC to prevent peptide bond formation between the amino acids Ile9 (P-site) and Ser10 (A-site) (Figure 1B). How the signal is transferred to the PTC and the mechanism of PTC inactivation are addressed by Ramu et al. (2011) by comparing ErmCL stalling with another stalling peptide present in the ermA operon. The ermA gene also encodes a methyltransferase that confers resistance to erythromycin, but unlike the ermC operon, two leader peptides, termed ermAL1 and ermAL2, are located upstream of the ermA gene (Ramu et al., 2009). Mankin and coworkers show that while the ErmAL2 stalling sequence operates analogous to ErmCL, the sequence (Figure 1A) and stalling mechanism of the ErmAL1 peptide differs: Translation of ermAL1 stalls when the eighth codon (Val8) is located in the P-site; however, unlike ErmCL stalling, the identity of the A-site (ninth) amino acid dramatically affects the efficiency of stalling (Ramu et al., 2011). ErmAL1 stalling is most efficient when the ninth amino acid is charged, such as Asp, Lys, Arg, His, or the native Glu, but also occurs with uncharged amino acids Trp, Tyr, and Ile
(Figure 1C). In contrast, when Phe, Met, or Cys is at the ninth position of the ErmAL1 peptide, translational stalling is reduced or abolished (Ramu et al., 2011). Generating chimeric ErmCL-AL1 peptides revealed that the 2 position (Phe7 in ErmCL or Ala6 in ErmAL1) regulates whether the PTC is selective, by allowing incorporation of a subset of amino acids (as in ErmAL1) (Figure 1C), or restrictive, i.e., by preventing incorporation of all amino acids (as in ErmCL [Figure 1D]). The proximity of the 2 position of the Erm peptides to A2062, A2503, and the cladinose of erythromycin led Mankin and coworkers to propose two relays for inactivation of the A-site of the PTC, one operating through U2504 and C2452 and the other through G2061 and A2451 (Ramu et al., 2011) (Figure 1E). A2451 and C2452 form a cleft into which the aminoacyl moiety of the A-tRNA is positioned for peptide bond formation (Figure 1E); therefore, it is easy to envisage how slight alterations in the position of these nucleotides could generate a narrower restrictive or wider selective A-site cleft. With these new insights into the mechanism of action of the Erm peptides in hand, one important future direction will be to assess how generally applicable the results are to other nascent peptide-mediated stalling systems, including those that are drug independent. An obvious candidate is the SecM leader peptide, where translational arrest is also abolished by mutation of the 2 position (Arg163) of the SecM peptide (Yap and Bernstein, 2009) as well as by mutations of A2062 or A2503 of the 23S rRNA (Va´zquez-Laslop et al., 2010). Moreover, a putative contact between A2062 and Arg163 of SecM is observed in the cryoelectron microscopy
Molecular Cell 41, February 4, 2011 ª2011 Elsevier Inc. 247
Molecular Cell
Previews
Laslop et al., 2010), indicating that the relay mechanism of TnaC is likely to be distinct from ErmCL and SecM. Indeed, stalling of other Erm peptides, such as ErmBL and ErmDL, is also independent of A2062/A2503 (Va´zquez-Laslop et al., 2010). In addition, many stalling sequences are longer than the Erm peptides, with critical residues ( 9 to 12 positions) being located deeper in the tunnel (Bhushan et al., 2010, 2011; Cruz-Vera et al., 2005; Seidelt et al., 2009; Yap and Bernstein, 2009). This deeper region of the tunnel is constricted and represents a hot spot for mutations that alleviate translational arrest (Ito et al., 2010). Thus, from the relatively small number of stalling peptides that have so far been identified and characterized, it appears likely that a diverse array of allosteric mechanisms are employed by distinct stalling peptides to attain the common goal: namely, translational arrest. REFERENCES Bhushan, S., Meyer, H., Starosta, A.L., Becker, T., Mielke, T., Berninghausen, O., Sattler, M., Wilson, D.N., and Beckmann, R. (2010). Mol. Cell 40, 138–146.
Figure 1. Mechanistic Insights into ErmCL- and ErmAL-Mediated Translational Arrest (A) In the absence of erythromycin, stem combinations 1 + 2 and 3 + 4 are favored, leading to repression of ermC due to the sequestering of the ribosome-binding site (RBS) and AUG start codon of ermC within the 3 + 4 stem loop. In the presence of subinhibitory concentrations of erythromycin, stalling of the ribosome at the ninth codon (Ile9) during translation of ermCL leads to an alternative 2 + 3 stem loop, allowing ribosomes to access the RBS and AUG of the ermC gene. In contrast, translational stalling of ErmAL1 occurs when the eighth codon (Val8) is in the P-site. (B) Schematic of the ErmCL stalling ribosome, with ErmCL-tRNAIle in the P-site (gold) and the tenth codon encoding Ser-tRNASer located in the A-site. Interaction between erythromycin (blue), the ErmCL peptide, and the ribosome relays a signal (arrowed) back to inactivate the peptidyl transferase center (PTC). (C) In the presence of erythromycin (Ery) and ErmAL1 (Ala is 2 position), the A-site of the peptidyl transferase center (PTC) becomes selective: Peptide bond formation with certain amino acids is abolished (red) or becomes very slow (orange), leading to stalling or partial stalling, but other amino acids are permitted (green), and no stalling ensues. (D) In contrast, ErmCL (Phe at 2 position) renders the A-site restrictive, preventing all amino acids from forming peptide bonds, leading to translational stalling. (E) Two possible signal-relay pathways (arrowed) communicating the information from the ribosomal exit tunnel to the PTC A-site. Erythromycin, blue; ErmAL-tRNA in the P-site (yellow) has the 2 position colored red. The aminoacyl moiety of the A-site tRNA (gray) inserts into the cleft between A2451 and A2452 (light blue) of the 23S rRNA. Other potential nucleotides involved in the relays include A2503 and U2504 (cyan) as well as A2062 and G2061 (teal).
structure of a SecM-stalled ribosome (Bhushan et al., 2011). Curiously, in this structure the CCA end of the SecM-tRNA is mispositioned at the P-site of the PTC (Bhushan et al., 2011), raising the question of whether a similar shift in the Erm-tRNAs also contributes to Erm peptide-mediated arrest. While A-site selectivity has also been observed with the TnaC stalling
peptide (Cruz-Vera et al., 2005), sequence conservation as well as biochemical (CruzVera and Yanofsky, 2008) and structural data (Seidelt et al., 2009) would suggest it is the 3 position (Asp21), rather than 2 position, that is important for arrest. However, it should be noted that mutation of A2062 or A2503 does not abolish TnaCmediated translational stalling (Va´zquez-
248 Molecular Cell 41, February 4, 2011 ª2011 Elsevier Inc.
Bhushan, S., Hoffmann, T., Seidelt, B., Frauenfeld, J., Mielke, T., Berninghausen, O., Wilson, D.N., and Beckmann, R. (2011). PLoS Biol., in press. Published online January 18, 2011. 10.1371/journal. pbio.1000581. Cruz-Vera, L.R., and Yanofsky, C. (2008). J. Bacteriol. 190, 4791–4797. Cruz-Vera, L.R., Rajagopal, S., Squires, C., and Yanofsky, C. (2005). Mol. Cell 19, 333–343. Ito, K., Chiba, S., and Pogliano, K. (2010). Biochem. Biophys. Res. Commun. 393, 1–5. Ramu, H., Mankin, A., and Vazquez-Laslop, N. (2009). Mol. Microbiol. 71, 811–824. Ramu, H., Vazquez-Laslop, N., Klepacki, D., Dai, Q., Piccirilli, J., Micura, R., and Mankin, A. (2011). Mol. Cell 41, this issue, 321–330. Seidelt, B., Innis, C.A., Wilson, D.N., Gartmann, M., Armache, J.P., Villa, E., Trabuco, L.G., Becker, T., Mielke, T., Schulten, K., et al. (2009). Science 326, 1412–1415. Va´zquez-Laslop, N., Thum, C., and Mankin, A.S. (2008). Mol. Cell 30, 190–202. Va´zquez-Laslop, N., Ramu, H., Klepacki, D., Kannan, K., and Mankin, A.S. (2010). EMBO J. 29, 3108–3117. Yap, M.N., and Bernstein, H.D. (2009). Mol. Cell 34, 201–211.