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

Neubauer C, Gao YG, Andersen KR, Dunham CM, Kelley AC, Hentschel J, Gerdes K, Ramakrishnan V, Brodersen DE - Cell (2009)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: MRC Laboratory of Molecular Biology, Cambridge CB2 0QH, UK.

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.

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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 pKa 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.
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fig5: 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 pKa 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.

Mentions: To clarify the involvement of the individual residues in catalysis, we constructed K52A, K54A, R61A, R81A, Y87A, and Y87F mutants of RelE and measured their cleavage efficiencies on T. thermophilus 70S ribosomes in vitro (Figure 5A). Based on our structures, we additionally designed a R81A/Y87F double mutant that we expected to have the strongest effect. Neither the lysine (K52A, K54A) nor the Y87F mutants showed a significant decrease in activity. However, mutation of either of the arginines (R61A, R81A) or the tyrosine (Y87A) significantly lowered the cleavage efficiency. Most remarkably, the R81A/Y87F double mutant almost completely abolished RelE activity. Together, these data indicate that both the arginines and the stacking of Y87 with the mRNA are important for the cleavage reaction and that the loss of the Y87 η-OH group can be compensated only when R81 is retained.


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

Neubauer C, Gao YG, Andersen KR, Dunham CM, Kelley AC, Hentschel J, Gerdes K, Ramakrishnan V, Brodersen DE - Cell (2009)

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 pKa 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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2807027&req=5

fig5: 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 pKa 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.
Mentions: To clarify the involvement of the individual residues in catalysis, we constructed K52A, K54A, R61A, R81A, Y87A, and Y87F mutants of RelE and measured their cleavage efficiencies on T. thermophilus 70S ribosomes in vitro (Figure 5A). Based on our structures, we additionally designed a R81A/Y87F double mutant that we expected to have the strongest effect. Neither the lysine (K52A, K54A) nor the Y87F mutants showed a significant decrease in activity. However, mutation of either of the arginines (R61A, R81A) or the tyrosine (Y87A) significantly lowered the cleavage efficiency. Most remarkably, the R81A/Y87F double mutant almost completely abolished RelE activity. Together, these data indicate that both the arginines and the stacking of Y87 with the mRNA are important for the cleavage reaction and that the loss of the Y87 η-OH group can be compensated only when R81 is retained.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: MRC Laboratory of Molecular Biology, Cambridge CB2 0QH, UK.

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.

Show MeSH
Related in: MedlinePlus