Limits...
YoeB-ribosome structure: a canonical RNase that requires the ribosome for its specific activity.

Feng S, Chen Y, Kamada K, Wang H, Tang K, Wang M, Gao YG - Nucleic Acids Res. (2013)

Bottom Line: Also, the mRNA orientation involves the universally conserved A1493 and G530 of 16S rRNA.In addition, mass spectrometry data indicated that YoeB cleaves mRNA following the second position at the A-site codon, resulting in a final product with a 3'-phosphate at the newly formed 3' end.Our results demonstrate a classical acid-base catalysis for YoeB-mediated RNA hydrolysis and provide insight into how the ribosome is essential for its specific activity.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Science, Nanyang Technological University, 637551 Singapore, RIKEN Advanced Science Institute, Saitama 351-0198, Japan, Swiss Light Source, Paul Scherrer Institut, CH-5232, Switzerland and Institute of Molecular and Cell Biology, A-STAR, 138673, Singapore.

ABSTRACT
As a typical endoribonuclease, YoeB mediates cellular adaptation in diverse bacteria by degrading mRNAs on its activation. Although the catalytic core of YoeB is thought to be identical to well-studied nucleases, this enzyme specifically targets mRNA substrates that are associated with ribosomes in vivo. However, the molecular mechanism of mRNA recognition and cleavage by YoeB, and the requirement of ribosome for its optimal activity, largely remain elusive. Here, we report the structure of YoeB bound to 70S ribosome in pre-cleavage state, revealing that both the 30S and 50S subunits participate in YoeB binding. The mRNA is recognized by the catalytic core of YoeB, of which the general base/acid (Glu46/His83) are within hydrogen-bonding distance to their reaction atoms, demonstrating an active conformation of YoeB on ribosome. Also, the mRNA orientation involves the universally conserved A1493 and G530 of 16S rRNA. In addition, mass spectrometry data indicated that YoeB cleaves mRNA following the second position at the A-site codon, resulting in a final product with a 3'-phosphate at the newly formed 3' end. Our results demonstrate a classical acid-base catalysis for YoeB-mediated RNA hydrolysis and provide insight into how the ribosome is essential for its specific activity.

Show MeSH
Interactions at the catalytic site (A) Interactions of YoeB with ribosome at the decoding center. The mRNA is shown as ribbon colored magenta. The residues in YoeB within hydrogen-bonding distance to ribosome are indicated by dashed lines. (B) Active site of YoeB surrounded by the A-site codon. Catalytic residues Glu46 and His83 are within hydrogen-bonding distance to their reaction atoms, indicated by dashed lines. Compared with isolated YoeB colored gray, conformational change of C-terminal tail of YoeB (particularly Tyr84) was shown by an arrow.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3814384&req=5

gkt742-F4: Interactions at the catalytic site (A) Interactions of YoeB with ribosome at the decoding center. The mRNA is shown as ribbon colored magenta. The residues in YoeB within hydrogen-bonding distance to ribosome are indicated by dashed lines. (B) Active site of YoeB surrounded by the A-site codon. Catalytic residues Glu46 and His83 are within hydrogen-bonding distance to their reaction atoms, indicated by dashed lines. Compared with isolated YoeB colored gray, conformational change of C-terminal tail of YoeB (particularly Tyr84) was shown by an arrow.

Mentions: In contrast to monomer B, monomer A forms extensive contacts with rRNAs (both 16S and 23S), ribosomal protein S12 and mRNA (Figures 2C and 3B). The N-terminal α1 and α2 form a V-shaped arrangement wedging into the cleft between the head and body of 30S, where the basic residues Arg22 and Lys26 interact with U531 and C519 in the decoding region (Figures 2C and 3B). On the opposite side of monomer A, it forms strong interactions with the tip of H69 of 23S rRNA, A1492/A1493 in h44 of 16S rRNA, and S12 (Figures 3B and 4A). The residues Thr71 and Asp72, located in the β-strand loop, establish hydrogen-bonding interactions with U1915 and C1914 in H69. Additionally, Phe39 enhances the interaction of YoeB with H69 via hydrophobic contact (A1913). H69, a highly conserved stem-loop (1906–1924) involved in the formation of intersubunit bridge B2a, is capable of interacting with translational factors such as release factors RF1 and RF2 (21,22), indicating a pivotal role in translation (23). YoeB tightly contacts H69, thereby likely interfering with its function. The neighbouring residue Glu40 of YoeB interacts with Thr44 located at the highly conserved β-loop of S12, which projects into the decoding center to participate in codon–anticodon recognition (24). In proximity, Lys42 forms bilateral contacts with ribose O3 of C519 in h18 of 16S rRNA and Leu52 in the β-loop of S12, via hydrogen bond and side-chain hydrophobic interaction, respectively (Figure 3B). Moreover, the main chain N atom of Gly43 contacts phosphate oxygen of C519 by ∼3.0 Å. In particular, a network of interactions involving YoeB, G530, S12, and mRNA is formed, of which Lys44 in YoeB interacts with the highly conserved Pro48 in S12 (Figure 4A). Pro48 is crucial for ribosome function, as mutation of the equivalent residue in E. coli S12 resulted in severe dominant growth defects (25).Figure 4.


YoeB-ribosome structure: a canonical RNase that requires the ribosome for its specific activity.

Feng S, Chen Y, Kamada K, Wang H, Tang K, Wang M, Gao YG - Nucleic Acids Res. (2013)

Interactions at the catalytic site (A) Interactions of YoeB with ribosome at the decoding center. The mRNA is shown as ribbon colored magenta. The residues in YoeB within hydrogen-bonding distance to ribosome are indicated by dashed lines. (B) Active site of YoeB surrounded by the A-site codon. Catalytic residues Glu46 and His83 are within hydrogen-bonding distance to their reaction atoms, indicated by dashed lines. Compared with isolated YoeB colored gray, conformational change of C-terminal tail of YoeB (particularly Tyr84) was shown by an arrow.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814384&req=5

gkt742-F4: Interactions at the catalytic site (A) Interactions of YoeB with ribosome at the decoding center. The mRNA is shown as ribbon colored magenta. The residues in YoeB within hydrogen-bonding distance to ribosome are indicated by dashed lines. (B) Active site of YoeB surrounded by the A-site codon. Catalytic residues Glu46 and His83 are within hydrogen-bonding distance to their reaction atoms, indicated by dashed lines. Compared with isolated YoeB colored gray, conformational change of C-terminal tail of YoeB (particularly Tyr84) was shown by an arrow.
Mentions: In contrast to monomer B, monomer A forms extensive contacts with rRNAs (both 16S and 23S), ribosomal protein S12 and mRNA (Figures 2C and 3B). The N-terminal α1 and α2 form a V-shaped arrangement wedging into the cleft between the head and body of 30S, where the basic residues Arg22 and Lys26 interact with U531 and C519 in the decoding region (Figures 2C and 3B). On the opposite side of monomer A, it forms strong interactions with the tip of H69 of 23S rRNA, A1492/A1493 in h44 of 16S rRNA, and S12 (Figures 3B and 4A). The residues Thr71 and Asp72, located in the β-strand loop, establish hydrogen-bonding interactions with U1915 and C1914 in H69. Additionally, Phe39 enhances the interaction of YoeB with H69 via hydrophobic contact (A1913). H69, a highly conserved stem-loop (1906–1924) involved in the formation of intersubunit bridge B2a, is capable of interacting with translational factors such as release factors RF1 and RF2 (21,22), indicating a pivotal role in translation (23). YoeB tightly contacts H69, thereby likely interfering with its function. The neighbouring residue Glu40 of YoeB interacts with Thr44 located at the highly conserved β-loop of S12, which projects into the decoding center to participate in codon–anticodon recognition (24). In proximity, Lys42 forms bilateral contacts with ribose O3 of C519 in h18 of 16S rRNA and Leu52 in the β-loop of S12, via hydrogen bond and side-chain hydrophobic interaction, respectively (Figure 3B). Moreover, the main chain N atom of Gly43 contacts phosphate oxygen of C519 by ∼3.0 Å. In particular, a network of interactions involving YoeB, G530, S12, and mRNA is formed, of which Lys44 in YoeB interacts with the highly conserved Pro48 in S12 (Figure 4A). Pro48 is crucial for ribosome function, as mutation of the equivalent residue in E. coli S12 resulted in severe dominant growth defects (25).Figure 4.

Bottom Line: Also, the mRNA orientation involves the universally conserved A1493 and G530 of 16S rRNA.In addition, mass spectrometry data indicated that YoeB cleaves mRNA following the second position at the A-site codon, resulting in a final product with a 3'-phosphate at the newly formed 3' end.Our results demonstrate a classical acid-base catalysis for YoeB-mediated RNA hydrolysis and provide insight into how the ribosome is essential for its specific activity.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Science, Nanyang Technological University, 637551 Singapore, RIKEN Advanced Science Institute, Saitama 351-0198, Japan, Swiss Light Source, Paul Scherrer Institut, CH-5232, Switzerland and Institute of Molecular and Cell Biology, A-STAR, 138673, Singapore.

ABSTRACT
As a typical endoribonuclease, YoeB mediates cellular adaptation in diverse bacteria by degrading mRNAs on its activation. Although the catalytic core of YoeB is thought to be identical to well-studied nucleases, this enzyme specifically targets mRNA substrates that are associated with ribosomes in vivo. However, the molecular mechanism of mRNA recognition and cleavage by YoeB, and the requirement of ribosome for its optimal activity, largely remain elusive. Here, we report the structure of YoeB bound to 70S ribosome in pre-cleavage state, revealing that both the 30S and 50S subunits participate in YoeB binding. The mRNA is recognized by the catalytic core of YoeB, of which the general base/acid (Glu46/His83) are within hydrogen-bonding distance to their reaction atoms, demonstrating an active conformation of YoeB on ribosome. Also, the mRNA orientation involves the universally conserved A1493 and G530 of 16S rRNA. In addition, mass spectrometry data indicated that YoeB cleaves mRNA following the second position at the A-site codon, resulting in a final product with a 3'-phosphate at the newly formed 3' end. Our results demonstrate a classical acid-base catalysis for YoeB-mediated RNA hydrolysis and provide insight into how the ribosome is essential for its specific activity.

Show MeSH