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Crucial elements that maintain the interactions between the regulatory TnaC peptide and the ribosome exit tunnel responsible for Trp inhibition of ribosome function.

Martínez AK, Shirole NH, Murakami S, Benedik MJ, Sachs MS, Cruz-Vera LR - Nucleic Acids Res. (2011)

Bottom Line: Nucleotides A752 and U2609 establish a base-pair interaction.These data indicate that the TnaC nascent peptide in the ribosome exit tunnel interacts with the U2609 nucleotide when the ribosome is Trp responsive.This interaction is affected by mutational changes in exit tunnel nucleotides of 23S rRNA, as well as in conserved TnaC residues, suggesting that they affect the structure of the exit tunnel and/or the nascent peptide configuration in the tunnel.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Texas A&M University, College Station, TX 77843, USA.

ABSTRACT
Translation of the TnaC nascent peptide inhibits ribosomal activity in the presence of l-tryptophan, inducing expression of the tnaCAB operon in Escherichia coli. Using chemical methylation, this work reveals how interactions between TnaC and the ribosome are affected by mutations in both molecules. The presence of the TnaC-tRNA(Pro) peptidyl-tRNA within the ribosome protects the 23S rRNA nucleotide U2609 against chemical methylation. Such protection was not observed in mutant ribosomes containing changes in 23S rRNA nucleotides of the A748-A752 region. Nucleotides A752 and U2609 establish a base-pair interaction. Most replacements of either A752 or U2609 affected Trp induction of a TnaC-regulated LacZ reporter. However, the single change A752G, or the dual replacements A752G and U2609C, maintained Trp induction. Replacements at the conserved TnaC residues W12 and D16 also abolished the protection of U2609 by TnaC-tRNA(Pro) against chemical methylation. These data indicate that the TnaC nascent peptide in the ribosome exit tunnel interacts with the U2609 nucleotide when the ribosome is Trp responsive. This interaction is affected by mutational changes in exit tunnel nucleotides of 23S rRNA, as well as in conserved TnaC residues, suggesting that they affect the structure of the exit tunnel and/or the nascent peptide configuration in the tunnel.

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Regions of the ribosomal exit tunnel essential for stalling. (A) Lateral vision of the 70S ribosome of E. coli (20). (B) Sagittal plane section of the 70S ribosome (20). (C) Visual amplification of the PTC and the first part of the exit tunnel. Nucleotides in orange constitute the PTC region; these nucleotides are involved in the peptidyl transferase and hydrolysis of peptidyl-tRNAs during translation (38). Nucleotides in red are essential for stalling induced by the nascent peptides ErmCL and SecM (13,24). Nucleotides in pink and the amino acid residue K90 of the ribosomal protein L22 are essential for stalling induced by SecM and TnaC (13,19). Nucleotides in cyan connect the nucleotides U2585 and U2609. White arrow indicates possible structural relay from the exit tunnel to the PTC produced by the TnaC nascent peptide.
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gkr1052-F4: Regions of the ribosomal exit tunnel essential for stalling. (A) Lateral vision of the 70S ribosome of E. coli (20). (B) Sagittal plane section of the 70S ribosome (20). (C) Visual amplification of the PTC and the first part of the exit tunnel. Nucleotides in orange constitute the PTC region; these nucleotides are involved in the peptidyl transferase and hydrolysis of peptidyl-tRNAs during translation (38). Nucleotides in red are essential for stalling induced by the nascent peptides ErmCL and SecM (13,24). Nucleotides in pink and the amino acid residue K90 of the ribosomal protein L22 are essential for stalling induced by SecM and TnaC (13,19). Nucleotides in cyan connect the nucleotides U2585 and U2609. White arrow indicates possible structural relay from the exit tunnel to the PTC produced by the TnaC nascent peptide.

Mentions: The data presented here show that, in ribosomes that can respond to functional TnaC, functional TnaC-tRNAPro in the ribosome exit tunnel protects U2609 of the 23S rRNA from methylation by CMCT (Figure 2). These results are consistent with the structural model obtained from cryo-EM data, where it has been observed that U2609 conformation is affected by the presence of the TnaC peptide (21). The cryo-EM model suggests that the changes in the conformation of U2609 are not the result of major structural changes in the exit tunnel (21). These data suggest that interaction between U2609 and TnaC residues is a major reason for the difference in methylation sensitivity of U2609 when comparing ribosomes containing either no peptide or non-functional TnaC peptide to ribosomes containing functional TnaC peptide. The cryo-EM model further suggests that the K18 residue of TnaC might be involved in positioning U2609 (21). However, our mutagenesis analyses did not support this view. Replacing the K18 residue by alanine, a small non-charged amino acid, did not affect either Trp induction in vivo (Table 3) or inhibition of TnaC-tRNAPro cleavage by puromycin (Figure 3A). Also, the protection of U2609 was not affected by the K18A TnaC mutation (Figure 3B). The TnaC mutations W12R and D16A, which eliminate Trp-mediated ribosome stalling (Table 3 and Figure 3A), abolished the protection from methylation of U2609 (Figure 3B). Therefore, these essential residues of the TnaC peptide may interact with U2609 directly. Alternatively, W12 and D16 interactions with other elements of the ribosome may relay structural changes through the TnaC peptide to establish a position for U2609 that protects this nucleotide from methylation (21,27). Cryo-EM structures of eukaryotic ribosomes containing either of the regulatory peptides CMV or AAP suggest that amino acids that are important for stalling interact with the A751 and U2609 nucleotides (28). The essential residues for stalling Ser-12 of CMV and the Asp-12 of AAP seem to be in the proximity of A751 (18,28,29). Meanwhile, the important residues Lys-18 of CMV and Trp-19 of AAP are close to U2609 (18,28,29). These positions correspond with the conserved residues W12 and Ile-19 of TnaC (8). Furthermore, comparison of the cryo-EM structures of ribosomes containing each of these regulatory peptides reveal that they interact in similar manner with the A751–752 and U2609 region (43). These observations suggest that the interactions between the region constituted by A751–A752 and U2609 and residues of regulatory peptides are essential for stalling in prokaryotic and eukaryotic systems. This region of the ribosomal exit tunnel might be a common anchor-place for regulatory peptides (Figure 4). The ErmCL peptide seems to be the exception as the action of this regulatory peptide is not affected by mutations in the A751 and U2609 nucleotides (24). The ErmCL peptide is shorter and might not reach these nucleotides, in fact ErmCL is anchored to the A2058 nucleotide by erythromycin (Figure 4) (15).Figure 4.


Crucial elements that maintain the interactions between the regulatory TnaC peptide and the ribosome exit tunnel responsible for Trp inhibition of ribosome function.

Martínez AK, Shirole NH, Murakami S, Benedik MJ, Sachs MS, Cruz-Vera LR - Nucleic Acids Res. (2011)

Regions of the ribosomal exit tunnel essential for stalling. (A) Lateral vision of the 70S ribosome of E. coli (20). (B) Sagittal plane section of the 70S ribosome (20). (C) Visual amplification of the PTC and the first part of the exit tunnel. Nucleotides in orange constitute the PTC region; these nucleotides are involved in the peptidyl transferase and hydrolysis of peptidyl-tRNAs during translation (38). Nucleotides in red are essential for stalling induced by the nascent peptides ErmCL and SecM (13,24). Nucleotides in pink and the amino acid residue K90 of the ribosomal protein L22 are essential for stalling induced by SecM and TnaC (13,19). Nucleotides in cyan connect the nucleotides U2585 and U2609. White arrow indicates possible structural relay from the exit tunnel to the PTC produced by the TnaC nascent peptide.
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gkr1052-F4: Regions of the ribosomal exit tunnel essential for stalling. (A) Lateral vision of the 70S ribosome of E. coli (20). (B) Sagittal plane section of the 70S ribosome (20). (C) Visual amplification of the PTC and the first part of the exit tunnel. Nucleotides in orange constitute the PTC region; these nucleotides are involved in the peptidyl transferase and hydrolysis of peptidyl-tRNAs during translation (38). Nucleotides in red are essential for stalling induced by the nascent peptides ErmCL and SecM (13,24). Nucleotides in pink and the amino acid residue K90 of the ribosomal protein L22 are essential for stalling induced by SecM and TnaC (13,19). Nucleotides in cyan connect the nucleotides U2585 and U2609. White arrow indicates possible structural relay from the exit tunnel to the PTC produced by the TnaC nascent peptide.
Mentions: The data presented here show that, in ribosomes that can respond to functional TnaC, functional TnaC-tRNAPro in the ribosome exit tunnel protects U2609 of the 23S rRNA from methylation by CMCT (Figure 2). These results are consistent with the structural model obtained from cryo-EM data, where it has been observed that U2609 conformation is affected by the presence of the TnaC peptide (21). The cryo-EM model suggests that the changes in the conformation of U2609 are not the result of major structural changes in the exit tunnel (21). These data suggest that interaction between U2609 and TnaC residues is a major reason for the difference in methylation sensitivity of U2609 when comparing ribosomes containing either no peptide or non-functional TnaC peptide to ribosomes containing functional TnaC peptide. The cryo-EM model further suggests that the K18 residue of TnaC might be involved in positioning U2609 (21). However, our mutagenesis analyses did not support this view. Replacing the K18 residue by alanine, a small non-charged amino acid, did not affect either Trp induction in vivo (Table 3) or inhibition of TnaC-tRNAPro cleavage by puromycin (Figure 3A). Also, the protection of U2609 was not affected by the K18A TnaC mutation (Figure 3B). The TnaC mutations W12R and D16A, which eliminate Trp-mediated ribosome stalling (Table 3 and Figure 3A), abolished the protection from methylation of U2609 (Figure 3B). Therefore, these essential residues of the TnaC peptide may interact with U2609 directly. Alternatively, W12 and D16 interactions with other elements of the ribosome may relay structural changes through the TnaC peptide to establish a position for U2609 that protects this nucleotide from methylation (21,27). Cryo-EM structures of eukaryotic ribosomes containing either of the regulatory peptides CMV or AAP suggest that amino acids that are important for stalling interact with the A751 and U2609 nucleotides (28). The essential residues for stalling Ser-12 of CMV and the Asp-12 of AAP seem to be in the proximity of A751 (18,28,29). Meanwhile, the important residues Lys-18 of CMV and Trp-19 of AAP are close to U2609 (18,28,29). These positions correspond with the conserved residues W12 and Ile-19 of TnaC (8). Furthermore, comparison of the cryo-EM structures of ribosomes containing each of these regulatory peptides reveal that they interact in similar manner with the A751–752 and U2609 region (43). These observations suggest that the interactions between the region constituted by A751–A752 and U2609 and residues of regulatory peptides are essential for stalling in prokaryotic and eukaryotic systems. This region of the ribosomal exit tunnel might be a common anchor-place for regulatory peptides (Figure 4). The ErmCL peptide seems to be the exception as the action of this regulatory peptide is not affected by mutations in the A751 and U2609 nucleotides (24). The ErmCL peptide is shorter and might not reach these nucleotides, in fact ErmCL is anchored to the A2058 nucleotide by erythromycin (Figure 4) (15).Figure 4.

Bottom Line: Nucleotides A752 and U2609 establish a base-pair interaction.These data indicate that the TnaC nascent peptide in the ribosome exit tunnel interacts with the U2609 nucleotide when the ribosome is Trp responsive.This interaction is affected by mutational changes in exit tunnel nucleotides of 23S rRNA, as well as in conserved TnaC residues, suggesting that they affect the structure of the exit tunnel and/or the nascent peptide configuration in the tunnel.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Texas A&M University, College Station, TX 77843, USA.

ABSTRACT
Translation of the TnaC nascent peptide inhibits ribosomal activity in the presence of l-tryptophan, inducing expression of the tnaCAB operon in Escherichia coli. Using chemical methylation, this work reveals how interactions between TnaC and the ribosome are affected by mutations in both molecules. The presence of the TnaC-tRNA(Pro) peptidyl-tRNA within the ribosome protects the 23S rRNA nucleotide U2609 against chemical methylation. Such protection was not observed in mutant ribosomes containing changes in 23S rRNA nucleotides of the A748-A752 region. Nucleotides A752 and U2609 establish a base-pair interaction. Most replacements of either A752 or U2609 affected Trp induction of a TnaC-regulated LacZ reporter. However, the single change A752G, or the dual replacements A752G and U2609C, maintained Trp induction. Replacements at the conserved TnaC residues W12 and D16 also abolished the protection of U2609 by TnaC-tRNA(Pro) against chemical methylation. These data indicate that the TnaC nascent peptide in the ribosome exit tunnel interacts with the U2609 nucleotide when the ribosome is Trp responsive. This interaction is affected by mutational changes in exit tunnel nucleotides of 23S rRNA, as well as in conserved TnaC residues, suggesting that they affect the structure of the exit tunnel and/or the nascent peptide configuration in the tunnel.

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Related in: MedlinePlus