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Base methylations in the double-stranded RNA by a fused methyltransferase bearing unwinding activity.

Kimura S, Ikeuchi Y, Kitahara K, Sakaguchi Y, Suzuki T, Suzuki T - Nucleic Acids Res. (2011)

Bottom Line: The N-terminal RlmL activity for m(2)G2445 formation was significantly enhanced by the C-terminal RlmK.Moreover, RlmKL had an unwinding activity of Helix 74, facilitating cooperative methylations of m(7)G2069 and m(2)G2445 during biogenesis of 50S subunit.In fact, we observed that RlmKL was involved in the efficient assembly of 50S subunit in a mutant strain lacking an RNA helicase deaD.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.

ABSTRACT
Modifications of rRNAs are clustered in functional regions of the ribosome. In Helix 74 of Escherichia coli 23S rRNA, guanosines at positions 2069 and 2445 are modified to 7-methylguanosine(m(7)G) and N(2)-methylguanosine(m(2)G), respectively. We searched for the gene responsible for m(7)G2069 formation, and identified rlmL, which encodes the methyltransferase for m(2)G2445, as responsible for the biogenesis of m(7)G2069. In vitro methylation of rRNA revealed that rlmL encodes a fused methyltransferase responsible for forming both m(7)G2069 and m(2)G2445. We renamed the gene rlmKL. The N-terminal RlmL activity for m(2)G2445 formation was significantly enhanced by the C-terminal RlmK. Moreover, RlmKL had an unwinding activity of Helix 74, facilitating cooperative methylations of m(7)G2069 and m(2)G2445 during biogenesis of 50S subunit. In fact, we observed that RlmKL was involved in the efficient assembly of 50S subunit in a mutant strain lacking an RNA helicase deaD.

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Tertiary and secondary structures of the E. coli 23S rRNA domain V and enzymatic formation of 7-methylguanosine (m7G2069) and N2-methylguanosine (m2G2445). (A) The tertiary structure of domain V in E. coli 23S rRNA (PDB ID 2aw4). The strands G2061-C2084, G2235-C2258, C2422-D2449 are colored blue, green and red, respectively. Coordinates for Helix 93 are not displayed because it lies in front of Helix 74. The silhouette of 50S subunit was showed in gray line. (B) The secondary structure of domain V in the E. coli 23S rRNA with modified nucleotides, 7-methylguanosine (m7G), N2-methylguanosine (m2G), 2′-O-methylguanosine (Gm), dihydrouridine (D), pseudouridine (Ψ), 2′-O-methylcytidine (Cm), 5-hydroxycytidine (ho5C), N2-methyladenosine (m2A) and 2′-O-methyluridine (Um). The numbers of the RNA helices and nucleotides are indicated. Watson–Crick-type base pairs are shown by bars, and wobble base pairs by black dots. Helix 74 and surrounding regions are boxed on the left. The color code for each strand is the same as (A). (C) Enzymatic formation of m7G2069 and m2G2445 is catalyzed by RlmK and RlmL, respectively, using Ado-Met as a methyl-group donor. Ado-Met is converted to Ado-Hcy (S-adenosylhomocysteine) by the reaction.
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gkr1287-F1: Tertiary and secondary structures of the E. coli 23S rRNA domain V and enzymatic formation of 7-methylguanosine (m7G2069) and N2-methylguanosine (m2G2445). (A) The tertiary structure of domain V in E. coli 23S rRNA (PDB ID 2aw4). The strands G2061-C2084, G2235-C2258, C2422-D2449 are colored blue, green and red, respectively. Coordinates for Helix 93 are not displayed because it lies in front of Helix 74. The silhouette of 50S subunit was showed in gray line. (B) The secondary structure of domain V in the E. coli 23S rRNA with modified nucleotides, 7-methylguanosine (m7G), N2-methylguanosine (m2G), 2′-O-methylguanosine (Gm), dihydrouridine (D), pseudouridine (Ψ), 2′-O-methylcytidine (Cm), 5-hydroxycytidine (ho5C), N2-methyladenosine (m2A) and 2′-O-methyluridine (Um). The numbers of the RNA helices and nucleotides are indicated. Watson–Crick-type base pairs are shown by bars, and wobble base pairs by black dots. Helix 74 and surrounding regions are boxed on the left. The color code for each strand is the same as (A). (C) Enzymatic formation of m7G2069 and m2G2445 is catalyzed by RlmK and RlmL, respectively, using Ado-Met as a methyl-group donor. Ado-Met is converted to Ado-Hcy (S-adenosylhomocysteine) by the reaction.

Mentions: In Helix 74, in domain V of the 23S rRNA, G2069 and G2445 are modified to form 7-methylguanosine (m7G) and N2-methylguanosine (m2G), respectively (Figure 1A and B). The methyltransferase RlmL, encoded by rlmL/ycbY, catalyzes S-adenosylmethionine (Ado-Met)-dependent m2G2445 formation (34). Although deletion of rlmL/ycbY resulted in a slight growth reduction phenotype, the functional and physiological role of m2G2445 remains unclear (34). The methyltransferase mediating the biogenesis of m7G2069 has not yet been identified. In this study, we employed a genome-wide screen of uncharacterized genes in E. coli using the ribonucleome analysis to search for the gene responsible for m7G2069 formation. We happened to identify rlmL/ycbY as responsible for the biogenesis of m7G2069. In fact, rlmL/ycbY encodes a fused methyltransferase with dual active sites responsible for forming both m7G2069 and m2G2445 (Figure 1C). Thus, rlmL/ycbY has been renamed rlmKL. Genetic and biochemical characterization of this unique enzyme has revealed that cooperative methylation of Helix 74 by RlmKL plays a key role in the efficient assembly of the 50S subunit.Figure 1.


Base methylations in the double-stranded RNA by a fused methyltransferase bearing unwinding activity.

Kimura S, Ikeuchi Y, Kitahara K, Sakaguchi Y, Suzuki T, Suzuki T - Nucleic Acids Res. (2011)

Tertiary and secondary structures of the E. coli 23S rRNA domain V and enzymatic formation of 7-methylguanosine (m7G2069) and N2-methylguanosine (m2G2445). (A) The tertiary structure of domain V in E. coli 23S rRNA (PDB ID 2aw4). The strands G2061-C2084, G2235-C2258, C2422-D2449 are colored blue, green and red, respectively. Coordinates for Helix 93 are not displayed because it lies in front of Helix 74. The silhouette of 50S subunit was showed in gray line. (B) The secondary structure of domain V in the E. coli 23S rRNA with modified nucleotides, 7-methylguanosine (m7G), N2-methylguanosine (m2G), 2′-O-methylguanosine (Gm), dihydrouridine (D), pseudouridine (Ψ), 2′-O-methylcytidine (Cm), 5-hydroxycytidine (ho5C), N2-methyladenosine (m2A) and 2′-O-methyluridine (Um). The numbers of the RNA helices and nucleotides are indicated. Watson–Crick-type base pairs are shown by bars, and wobble base pairs by black dots. Helix 74 and surrounding regions are boxed on the left. The color code for each strand is the same as (A). (C) Enzymatic formation of m7G2069 and m2G2445 is catalyzed by RlmK and RlmL, respectively, using Ado-Met as a methyl-group donor. Ado-Met is converted to Ado-Hcy (S-adenosylhomocysteine) by the reaction.
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Related In: Results  -  Collection

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gkr1287-F1: Tertiary and secondary structures of the E. coli 23S rRNA domain V and enzymatic formation of 7-methylguanosine (m7G2069) and N2-methylguanosine (m2G2445). (A) The tertiary structure of domain V in E. coli 23S rRNA (PDB ID 2aw4). The strands G2061-C2084, G2235-C2258, C2422-D2449 are colored blue, green and red, respectively. Coordinates for Helix 93 are not displayed because it lies in front of Helix 74. The silhouette of 50S subunit was showed in gray line. (B) The secondary structure of domain V in the E. coli 23S rRNA with modified nucleotides, 7-methylguanosine (m7G), N2-methylguanosine (m2G), 2′-O-methylguanosine (Gm), dihydrouridine (D), pseudouridine (Ψ), 2′-O-methylcytidine (Cm), 5-hydroxycytidine (ho5C), N2-methyladenosine (m2A) and 2′-O-methyluridine (Um). The numbers of the RNA helices and nucleotides are indicated. Watson–Crick-type base pairs are shown by bars, and wobble base pairs by black dots. Helix 74 and surrounding regions are boxed on the left. The color code for each strand is the same as (A). (C) Enzymatic formation of m7G2069 and m2G2445 is catalyzed by RlmK and RlmL, respectively, using Ado-Met as a methyl-group donor. Ado-Met is converted to Ado-Hcy (S-adenosylhomocysteine) by the reaction.
Mentions: In Helix 74, in domain V of the 23S rRNA, G2069 and G2445 are modified to form 7-methylguanosine (m7G) and N2-methylguanosine (m2G), respectively (Figure 1A and B). The methyltransferase RlmL, encoded by rlmL/ycbY, catalyzes S-adenosylmethionine (Ado-Met)-dependent m2G2445 formation (34). Although deletion of rlmL/ycbY resulted in a slight growth reduction phenotype, the functional and physiological role of m2G2445 remains unclear (34). The methyltransferase mediating the biogenesis of m7G2069 has not yet been identified. In this study, we employed a genome-wide screen of uncharacterized genes in E. coli using the ribonucleome analysis to search for the gene responsible for m7G2069 formation. We happened to identify rlmL/ycbY as responsible for the biogenesis of m7G2069. In fact, rlmL/ycbY encodes a fused methyltransferase with dual active sites responsible for forming both m7G2069 and m2G2445 (Figure 1C). Thus, rlmL/ycbY has been renamed rlmKL. Genetic and biochemical characterization of this unique enzyme has revealed that cooperative methylation of Helix 74 by RlmKL plays a key role in the efficient assembly of the 50S subunit.Figure 1.

Bottom Line: The N-terminal RlmL activity for m(2)G2445 formation was significantly enhanced by the C-terminal RlmK.Moreover, RlmKL had an unwinding activity of Helix 74, facilitating cooperative methylations of m(7)G2069 and m(2)G2445 during biogenesis of 50S subunit.In fact, we observed that RlmKL was involved in the efficient assembly of 50S subunit in a mutant strain lacking an RNA helicase deaD.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.

ABSTRACT
Modifications of rRNAs are clustered in functional regions of the ribosome. In Helix 74 of Escherichia coli 23S rRNA, guanosines at positions 2069 and 2445 are modified to 7-methylguanosine(m(7)G) and N(2)-methylguanosine(m(2)G), respectively. We searched for the gene responsible for m(7)G2069 formation, and identified rlmL, which encodes the methyltransferase for m(2)G2445, as responsible for the biogenesis of m(7)G2069. In vitro methylation of rRNA revealed that rlmL encodes a fused methyltransferase responsible for forming both m(7)G2069 and m(2)G2445. We renamed the gene rlmKL. The N-terminal RlmL activity for m(2)G2445 formation was significantly enhanced by the C-terminal RlmK. Moreover, RlmKL had an unwinding activity of Helix 74, facilitating cooperative methylations of m(7)G2069 and m(2)G2445 during biogenesis of 50S subunit. In fact, we observed that RlmKL was involved in the efficient assembly of 50S subunit in a mutant strain lacking an RNA helicase deaD.

Show MeSH
Related in: MedlinePlus