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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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Substrate recognition of RlmKL. (A) The secondary structure of transcript 3, which contains base flipping mutation C2096G/G2193C to be cut by MazF at the single position. Closed arrowhead show the cleavage position by MazF. (B) Design of mutants based on the transcript 3. Arrows and boxes indicate the substitutions. (C) Methylation activities of the transcript 3 variants. The left and right graphs show the end point of m7G2069 and m2G2445 formation, respectively. The relative activities of methylation (%) were calculated and normalized by wild-type sequence. (D) The recognized residues for m7G2069 and m2G2445 formation in transcript 3. The residues whose mutations reduced m7G2069 or m2G2445 formation to <50% are depicted as white letters. In the left panel, the residues that are depicted in blue background indicates the m7G2069 forming activity. In the right panel, the residues that are depicted in red background indicates the m2G2445 formation activity. Color gradation of blue and red background for each residue represents magnitude of importance for the methylation. (E) Crystal structure of Helix 74 and surrounding region in the E. coli 50S subunit. Coordinates were obtained from PBD (2aw4) (3). We added the methyl groups for m7G2069 and m2G2445, shown in red, respectively. The recognized residues for m7G2069 and m2G2445 formation are shown in blue and red, respectively.
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gkr1287-F6: Substrate recognition of RlmKL. (A) The secondary structure of transcript 3, which contains base flipping mutation C2096G/G2193C to be cut by MazF at the single position. Closed arrowhead show the cleavage position by MazF. (B) Design of mutants based on the transcript 3. Arrows and boxes indicate the substitutions. (C) Methylation activities of the transcript 3 variants. The left and right graphs show the end point of m7G2069 and m2G2445 formation, respectively. The relative activities of methylation (%) were calculated and normalized by wild-type sequence. (D) The recognized residues for m7G2069 and m2G2445 formation in transcript 3. The residues whose mutations reduced m7G2069 or m2G2445 formation to <50% are depicted as white letters. In the left panel, the residues that are depicted in blue background indicates the m7G2069 forming activity. In the right panel, the residues that are depicted in red background indicates the m2G2445 formation activity. Color gradation of blue and red background for each residue represents magnitude of importance for the methylation. (E) Crystal structure of Helix 74 and surrounding region in the E. coli 50S subunit. Coordinates were obtained from PBD (2aw4) (3). We added the methyl groups for m7G2069 and m2G2445, shown in red, respectively. The recognized residues for m7G2069 and m2G2445 formation are shown in blue and red, respectively.

Mentions: To identify the sites required for both methylations, we constructed a series of variants based on transcript 3 and measured the methylation capacity of each variant. The secondary structure of transcript 3, the detailed constructs of the variants and their methylation activities are shown in Figure 6A, B and C, respectively. The efficiency of methylation was measured as described in ‘Materials and Methods’ section (Supplementary Figure S7). The base flipping of Helix 80 reduced m7G2069 formation. In the P-loop, three variants, G2250C, G2251C and G2252C, lacked m7G2069 formation, and G2253C reduced m7G2069 formation, whereas the C2254A variant showed normal activity. All mutants in P-loop and H80 have efficient m2G2445 forming capacities. In the 12 nt ss region, three variants, C2427U, G2429A and A2430U showed reduced m7G2069 forming activity, whereas G2428C and G2429A lacked m2G2445 formation and C2427U and A2430U and 2431-2433 mutation reduced the m2G2445 formation. In Helix 74, decreased m2G2445 formation was detected in the base-flipping variant C2072G/G2437C. In addition, A2070G/U2441C, G2067C/C2443G and C2066G/G2444C mutants lacked the m2G2445 formation. The five base-flipping variants, A2071U/U2438A, A2070G/U2441, G2067C/C2443G, C2066G/G2444C, C2064G/G2446C and the base substitution mutant U2068A showed no m7G2069 forming activity. The decreased m7G2069 formation was observed in C2065G/G2445C mutant. No significant change in the methylation of the A2439G and C2440G variants of the internal loop of Helix 74 were observed.Figure 6.


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)

Substrate recognition of RlmKL. (A) The secondary structure of transcript 3, which contains base flipping mutation C2096G/G2193C to be cut by MazF at the single position. Closed arrowhead show the cleavage position by MazF. (B) Design of mutants based on the transcript 3. Arrows and boxes indicate the substitutions. (C) Methylation activities of the transcript 3 variants. The left and right graphs show the end point of m7G2069 and m2G2445 formation, respectively. The relative activities of methylation (%) were calculated and normalized by wild-type sequence. (D) The recognized residues for m7G2069 and m2G2445 formation in transcript 3. The residues whose mutations reduced m7G2069 or m2G2445 formation to <50% are depicted as white letters. In the left panel, the residues that are depicted in blue background indicates the m7G2069 forming activity. In the right panel, the residues that are depicted in red background indicates the m2G2445 formation activity. Color gradation of blue and red background for each residue represents magnitude of importance for the methylation. (E) Crystal structure of Helix 74 and surrounding region in the E. coli 50S subunit. Coordinates were obtained from PBD (2aw4) (3). We added the methyl groups for m7G2069 and m2G2445, shown in red, respectively. The recognized residues for m7G2069 and m2G2445 formation are shown in blue and red, respectively.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351187&req=5

gkr1287-F6: Substrate recognition of RlmKL. (A) The secondary structure of transcript 3, which contains base flipping mutation C2096G/G2193C to be cut by MazF at the single position. Closed arrowhead show the cleavage position by MazF. (B) Design of mutants based on the transcript 3. Arrows and boxes indicate the substitutions. (C) Methylation activities of the transcript 3 variants. The left and right graphs show the end point of m7G2069 and m2G2445 formation, respectively. The relative activities of methylation (%) were calculated and normalized by wild-type sequence. (D) The recognized residues for m7G2069 and m2G2445 formation in transcript 3. The residues whose mutations reduced m7G2069 or m2G2445 formation to <50% are depicted as white letters. In the left panel, the residues that are depicted in blue background indicates the m7G2069 forming activity. In the right panel, the residues that are depicted in red background indicates the m2G2445 formation activity. Color gradation of blue and red background for each residue represents magnitude of importance for the methylation. (E) Crystal structure of Helix 74 and surrounding region in the E. coli 50S subunit. Coordinates were obtained from PBD (2aw4) (3). We added the methyl groups for m7G2069 and m2G2445, shown in red, respectively. The recognized residues for m7G2069 and m2G2445 formation are shown in blue and red, respectively.
Mentions: To identify the sites required for both methylations, we constructed a series of variants based on transcript 3 and measured the methylation capacity of each variant. The secondary structure of transcript 3, the detailed constructs of the variants and their methylation activities are shown in Figure 6A, B and C, respectively. The efficiency of methylation was measured as described in ‘Materials and Methods’ section (Supplementary Figure S7). The base flipping of Helix 80 reduced m7G2069 formation. In the P-loop, three variants, G2250C, G2251C and G2252C, lacked m7G2069 formation, and G2253C reduced m7G2069 formation, whereas the C2254A variant showed normal activity. All mutants in P-loop and H80 have efficient m2G2445 forming capacities. In the 12 nt ss region, three variants, C2427U, G2429A and A2430U showed reduced m7G2069 forming activity, whereas G2428C and G2429A lacked m2G2445 formation and C2427U and A2430U and 2431-2433 mutation reduced the m2G2445 formation. In Helix 74, decreased m2G2445 formation was detected in the base-flipping variant C2072G/G2437C. In addition, A2070G/U2441C, G2067C/C2443G and C2066G/G2444C mutants lacked the m2G2445 formation. The five base-flipping variants, A2071U/U2438A, A2070G/U2441, G2067C/C2443G, C2066G/G2444C, C2064G/G2446C and the base substitution mutant U2068A showed no m7G2069 forming activity. The decreased m7G2069 formation was observed in C2065G/G2445C mutant. No significant change in the methylation of the A2439G and C2440G variants of the internal loop of Helix 74 were observed.Figure 6.

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