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Structure of the bifunctional methyltransferase YcbY (RlmKL) that adds the m7G2069 and m2G2445 modifications in Escherichia coli 23S rRNA.

Wang KT, Desmolaize B, Nan J, Zhang XW, Li LF, Douthwaite S, Su XD - Nucleic Acids Res. (2012)

Bottom Line: Smu776 does not target G2069, and this nucleotide remains unmodified in Gram-positive rRNAs.The E.coli YcbY enzyme is the first example of a methyltransferase catalyzing two mechanistically different types of RNA modification, and has been renamed as the Ribosomal large subunit methyltransferase, RlmKL.Our structural and functional data provide insights into how this bifunctional enzyme evolved.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Protein and Plant Gene Research, and Biodynamic Optical Imaging Center (BIOPIC), School of Life Sciences, Peking University, Beijing 100871, Republic of China.

ABSTRACT
The 23S rRNA nucleotide m(2)G2445 is highly conserved in bacteria, and in Escherichia coli this modification is added by the enzyme YcbY. With lengths of around 700 amino acids, YcbY orthologs are the largest rRNA methyltransferases identified in Gram-negative bacteria, and they appear to be fusions from two separate proteins found in Gram-positives. The crystal structures described here show that both the N- and C-terminal halves of E. coli YcbY have a methyltransferase active site and their folding patterns respectively resemble the Streptococcus mutans proteins Smu472 and Smu776. Mass spectrometric analyses of 23S rRNAs showed that the N-terminal region of YcbY and Smu472 are functionally equivalent and add the m(2)G2445 modification, while the C-terminal region of YcbY is responsible for the m(7)G2069 methylation on the opposite side of the same helix (H74). Smu776 does not target G2069, and this nucleotide remains unmodified in Gram-positive rRNAs. The E.coli YcbY enzyme is the first example of a methyltransferase catalyzing two mechanistically different types of RNA modification, and has been renamed as the Ribosomal large subunit methyltransferase, RlmKL. Our structural and functional data provide insights into how this bifunctional enzyme evolved.

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Crystal packing of YcbY as dimer. (A) The YcbY dimer in the asymmetric unit shown in ribbon mode. The upper N-terminal (red) and C-terminal halves (magenta) represent one monomer unit and interact in the crystal to the second (lower) monomer through binding of two n-octanoylsucrose detergent molecules (blue). SAH molecules (yellow) are shown in space filling mode. The intramolecular distances between the active sites (∼44 Å) are similar to the intermolecular distance between the sites in the dimer (∼40 Å). The consensus of data suggests that the monomeric form represents the true physiological state of YcbY. (B) Structure of the synthetic detergent n-octanoylsucrose.
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gks160-F5: Crystal packing of YcbY as dimer. (A) The YcbY dimer in the asymmetric unit shown in ribbon mode. The upper N-terminal (red) and C-terminal halves (magenta) represent one monomer unit and interact in the crystal to the second (lower) monomer through binding of two n-octanoylsucrose detergent molecules (blue). SAH molecules (yellow) are shown in space filling mode. The intramolecular distances between the active sites (∼44 Å) are similar to the intermolecular distance between the sites in the dimer (∼40 Å). The consensus of data suggests that the monomeric form represents the true physiological state of YcbY. (B) Structure of the synthetic detergent n-octanoylsucrose.

Mentions: In the assembled 50S structure, nucleotides G2069 and G2445 are folded within the subunit structure and appear inaccessible to the YcbY methyltransferase (48,49). Consistent with this, the methyltransferase has been shown to use naked 23S rRNA rather than assembled subunits as the substrate for both G2069 (47) and G2445 methylation (21,47). The distance spanning the two nucleotide targets through the minor groove of helix 74 is just over 10 Å, and thus well within the 44 Å distance between the active sites in the YcbY structure (Figure 5). The distance between the target nucleotides could be altered by opening of H74 through the helicase activity of the YcbY-C domain (47) and by flipping out the bases from a stacked conformation into the enzyme catalytic sites, as has been seen for other methyltransferases (50,51). Such changes in the rRNA substrate might be comfortably accommodated by domain movement around the flexible mid-section of YcbY (Supplementary Figure S7), making it feasible that recognition and modification of G2069 and G2445 occur through a single enzyme–rRNA-binding event.Figure 5.


Structure of the bifunctional methyltransferase YcbY (RlmKL) that adds the m7G2069 and m2G2445 modifications in Escherichia coli 23S rRNA.

Wang KT, Desmolaize B, Nan J, Zhang XW, Li LF, Douthwaite S, Su XD - Nucleic Acids Res. (2012)

Crystal packing of YcbY as dimer. (A) The YcbY dimer in the asymmetric unit shown in ribbon mode. The upper N-terminal (red) and C-terminal halves (magenta) represent one monomer unit and interact in the crystal to the second (lower) monomer through binding of two n-octanoylsucrose detergent molecules (blue). SAH molecules (yellow) are shown in space filling mode. The intramolecular distances between the active sites (∼44 Å) are similar to the intermolecular distance between the sites in the dimer (∼40 Å). The consensus of data suggests that the monomeric form represents the true physiological state of YcbY. (B) Structure of the synthetic detergent n-octanoylsucrose.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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gks160-F5: Crystal packing of YcbY as dimer. (A) The YcbY dimer in the asymmetric unit shown in ribbon mode. The upper N-terminal (red) and C-terminal halves (magenta) represent one monomer unit and interact in the crystal to the second (lower) monomer through binding of two n-octanoylsucrose detergent molecules (blue). SAH molecules (yellow) are shown in space filling mode. The intramolecular distances between the active sites (∼44 Å) are similar to the intermolecular distance between the sites in the dimer (∼40 Å). The consensus of data suggests that the monomeric form represents the true physiological state of YcbY. (B) Structure of the synthetic detergent n-octanoylsucrose.
Mentions: In the assembled 50S structure, nucleotides G2069 and G2445 are folded within the subunit structure and appear inaccessible to the YcbY methyltransferase (48,49). Consistent with this, the methyltransferase has been shown to use naked 23S rRNA rather than assembled subunits as the substrate for both G2069 (47) and G2445 methylation (21,47). The distance spanning the two nucleotide targets through the minor groove of helix 74 is just over 10 Å, and thus well within the 44 Å distance between the active sites in the YcbY structure (Figure 5). The distance between the target nucleotides could be altered by opening of H74 through the helicase activity of the YcbY-C domain (47) and by flipping out the bases from a stacked conformation into the enzyme catalytic sites, as has been seen for other methyltransferases (50,51). Such changes in the rRNA substrate might be comfortably accommodated by domain movement around the flexible mid-section of YcbY (Supplementary Figure S7), making it feasible that recognition and modification of G2069 and G2445 occur through a single enzyme–rRNA-binding event.Figure 5.

Bottom Line: Smu776 does not target G2069, and this nucleotide remains unmodified in Gram-positive rRNAs.The E.coli YcbY enzyme is the first example of a methyltransferase catalyzing two mechanistically different types of RNA modification, and has been renamed as the Ribosomal large subunit methyltransferase, RlmKL.Our structural and functional data provide insights into how this bifunctional enzyme evolved.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Protein and Plant Gene Research, and Biodynamic Optical Imaging Center (BIOPIC), School of Life Sciences, Peking University, Beijing 100871, Republic of China.

ABSTRACT
The 23S rRNA nucleotide m(2)G2445 is highly conserved in bacteria, and in Escherichia coli this modification is added by the enzyme YcbY. With lengths of around 700 amino acids, YcbY orthologs are the largest rRNA methyltransferases identified in Gram-negative bacteria, and they appear to be fusions from two separate proteins found in Gram-positives. The crystal structures described here show that both the N- and C-terminal halves of E. coli YcbY have a methyltransferase active site and their folding patterns respectively resemble the Streptococcus mutans proteins Smu472 and Smu776. Mass spectrometric analyses of 23S rRNAs showed that the N-terminal region of YcbY and Smu472 are functionally equivalent and add the m(2)G2445 modification, while the C-terminal region of YcbY is responsible for the m(7)G2069 methylation on the opposite side of the same helix (H74). Smu776 does not target G2069, and this nucleotide remains unmodified in Gram-positive rRNAs. The E.coli YcbY enzyme is the first example of a methyltransferase catalyzing two mechanistically different types of RNA modification, and has been renamed as the Ribosomal large subunit methyltransferase, RlmKL. Our structural and functional data provide insights into how this bifunctional enzyme evolved.

Show MeSH
Related in: MedlinePlus