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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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Unwinding activity of RlmKL. (A) The RNA substrate used for the unwinding assay. 5′-end of transcript 8 was labeled by 32P. (B) The unwinding assay by RlmKL. The duplex and single strands were separated by native PAGE and the radio activity of transcript 8 was visualized and quantified by the imaging analyzer. Duplex ratio (%) described at the bottom of the panel stands for the remaining duplex proportion in total radio activity of transcript 8. The left 5 lanes and the right 5 lanes show time course monitoring of the unwinding activity in the presence of RlmKL (RlmKL+) and BSA (BSA+), respectively. (C) Duplex ratio (%) was plotted against reaction time (min). Open and closed circles indicate BSA+ and RlmKL+, respectively. The half-lives (t1/2) for both conditions were calculated by non-linear curve fitting and shown as average ±SD; values of three independent experiments.
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gkr1287-F5: Unwinding activity of RlmKL. (A) The RNA substrate used for the unwinding assay. 5′-end of transcript 8 was labeled by 32P. (B) The unwinding assay by RlmKL. The duplex and single strands were separated by native PAGE and the radio activity of transcript 8 was visualized and quantified by the imaging analyzer. Duplex ratio (%) described at the bottom of the panel stands for the remaining duplex proportion in total radio activity of transcript 8. The left 5 lanes and the right 5 lanes show time course monitoring of the unwinding activity in the presence of RlmKL (RlmKL+) and BSA (BSA+), respectively. (C) Duplex ratio (%) was plotted against reaction time (min). Open and closed circles indicate BSA+ and RlmKL+, respectively. The half-lives (t1/2) for both conditions were calculated by non-linear curve fitting and shown as average ±SD; values of three independent experiments.

Mentions: To examine the unwinding activity of RlmKL, we performed the unwinding assay used for RNA helicases (36,37). The 5′-32P-labeled transcript 8 and non-labeled transcript 9 were annealed to prepare the duplex substrate for RlmKL (Figure 5A). The duplex and single strands were resolved by native PAGE to monitor the unwinding of the duplex with the passage of reaction time (Figure 5B). In the presence of RlmKL, 71% of the duplex substrate was unwound in the first 3 min, and 92% was unwound in 30 min (Figure 5B). As a control, in the presence of BSA, 29% of the duplex substrate was spontaneously converted to the single strand, and 66% was unwound in 30 min (Figure 5B). In the presence of RlmKL, the half-life (t1/2) of the duplex substrate was 2.5 min, whereas in the presence of BSA, t1/2 was 6.0 min (Figure 5C). We also examined the unwinding activity of RlmKL in the absence of Ado-Met, and found no change in the activity (data not shown). These results suggested that RlmKL has an activity to unwind Helix 74 during substrate recognition and methylation.Figure 5.


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)

Unwinding activity of RlmKL. (A) The RNA substrate used for the unwinding assay. 5′-end of transcript 8 was labeled by 32P. (B) The unwinding assay by RlmKL. The duplex and single strands were separated by native PAGE and the radio activity of transcript 8 was visualized and quantified by the imaging analyzer. Duplex ratio (%) described at the bottom of the panel stands for the remaining duplex proportion in total radio activity of transcript 8. The left 5 lanes and the right 5 lanes show time course monitoring of the unwinding activity in the presence of RlmKL (RlmKL+) and BSA (BSA+), respectively. (C) Duplex ratio (%) was plotted against reaction time (min). Open and closed circles indicate BSA+ and RlmKL+, respectively. The half-lives (t1/2) for both conditions were calculated by non-linear curve fitting and shown as average ±SD; values of three independent experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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gkr1287-F5: Unwinding activity of RlmKL. (A) The RNA substrate used for the unwinding assay. 5′-end of transcript 8 was labeled by 32P. (B) The unwinding assay by RlmKL. The duplex and single strands were separated by native PAGE and the radio activity of transcript 8 was visualized and quantified by the imaging analyzer. Duplex ratio (%) described at the bottom of the panel stands for the remaining duplex proportion in total radio activity of transcript 8. The left 5 lanes and the right 5 lanes show time course monitoring of the unwinding activity in the presence of RlmKL (RlmKL+) and BSA (BSA+), respectively. (C) Duplex ratio (%) was plotted against reaction time (min). Open and closed circles indicate BSA+ and RlmKL+, respectively. The half-lives (t1/2) for both conditions were calculated by non-linear curve fitting and shown as average ±SD; values of three independent experiments.
Mentions: To examine the unwinding activity of RlmKL, we performed the unwinding assay used for RNA helicases (36,37). The 5′-32P-labeled transcript 8 and non-labeled transcript 9 were annealed to prepare the duplex substrate for RlmKL (Figure 5A). The duplex and single strands were resolved by native PAGE to monitor the unwinding of the duplex with the passage of reaction time (Figure 5B). In the presence of RlmKL, 71% of the duplex substrate was unwound in the first 3 min, and 92% was unwound in 30 min (Figure 5B). As a control, in the presence of BSA, 29% of the duplex substrate was spontaneously converted to the single strand, and 66% was unwound in 30 min (Figure 5B). In the presence of RlmKL, the half-life (t1/2) of the duplex substrate was 2.5 min, whereas in the presence of BSA, t1/2 was 6.0 min (Figure 5C). We also examined the unwinding activity of RlmKL in the absence of Ado-Met, and found no change in the activity (data not shown). These results suggested that RlmKL has an activity to unwind Helix 74 during substrate recognition and methylation.Figure 5.

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