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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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Synthetic growth phenotype of ΔrlmKL and ribosomal subunit profiling. (A) Exploring the synthetic growth phenotype of ΔrlmKL in the presence of the disruption of RNA helicases, including deaD, rhlE, srmB and dbpA. Each strain was serially diluted (1:10 dilutions), spotted onto LB plates and incubated at 37°C or 28°C for 11 and 22 h, respectively. The doubling time (in minutes) is shown to the right of each panel. (B) SDG profiling of ribosomal subunits in cell lysates in the presence of 0.5 mM Mg2+ for the wild-type (blue line), ΔrlmKL (red line), ΔdeaD (red line) and ΔrlmKL/ΔdeaD (red line) strains. The amount of each subunit was quantified by UV absorbance at 254 nm. The peak positions for the 30S and 50S subunits are indicated. The peak height was normalized to the 30S peak.
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gkr1287-F7: Synthetic growth phenotype of ΔrlmKL and ribosomal subunit profiling. (A) Exploring the synthetic growth phenotype of ΔrlmKL in the presence of the disruption of RNA helicases, including deaD, rhlE, srmB and dbpA. Each strain was serially diluted (1:10 dilutions), spotted onto LB plates and incubated at 37°C or 28°C for 11 and 22 h, respectively. The doubling time (in minutes) is shown to the right of each panel. (B) SDG profiling of ribosomal subunits in cell lysates in the presence of 0.5 mM Mg2+ for the wild-type (blue line), ΔrlmKL (red line), ΔdeaD (red line) and ΔrlmKL/ΔdeaD (red line) strains. The amount of each subunit was quantified by UV absorbance at 254 nm. The peak positions for the 30S and 50S subunits are indicated. The peak height was normalized to the 30S peak.

Mentions: The protein product of ycbY has been reported to co-sediment with the early assembly intermediate of the 50S ribosomal subunit in E. coli (43). In addition, YcbY interacts with the DeaD and SrmB proteins, which are DEAD-box RNA helicases involved in 50S subunit formation (44–46). These results suggested that RlmKL may be involved in an early stage of the biogenesis of the 50S subunit. However, disruption of the rlmKL gene showed almost no growth phenotype even when cells were cultured at low temperature (Figure 7A), and no significant change in ribosome profile was detected (Figure 7B). Since YcbY interacts with DeaD and SrmB in E. coli (45), we explored the synthetic growth phenotype of the ΔrlmKL strain in which RNA helicases, including deaD, srmB, rhlE and dbpA, were knocked out. As shown in Figure 7A, we observed a synthetic growth phenotype with deaD, in particular at low temperature. When cultured at 37°C, the doubling time of the ΔrlmKL/ΔdeaD strain (28.4 min) was delayed by 4.5 min relative to the ΔrlmKL strain (23.9 min) and 2.9 min relative to the ΔdeaD strain (25.5 min). At 28°C, the ΔrlmKL/ΔdeaD strain exhibited a growth defect resulting in a doubling time of 99.0 min, a 50 min delay relative to the ΔrlmKL strain (49.0 min) and a 21.7 min delay relative to the ΔdeaD strain (77.3 min). This result suggested that RlmKL and the DeaD RNA helicase are cooperatively involved in efficient assembly of the 50S subunit. Therefore, the ribosome profile of the ΔrlmKL/ΔdeaD strain was analyzed by sucrose density gradient centrifugation. In the ΔdeaD strain (Figure 7B), a slight decrease in the 50S subunit compared with the wild-type strain, and an accumulation of a 40S fraction that contains an intermediate of the 50S subunit, were detected. In the ΔrlmKL/ΔdeaD strain (Figure 7B), an apparent decrease in the 50S subunit compared with the wild-type and ΔrlmKL strains and an accumulation of an intermediate 30–40S fraction were observed. These results indicated that deletion of rlmKL in the ΔdeaD background caused a considerable defect in 50S assembly.Figure 7.


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)

Synthetic growth phenotype of ΔrlmKL and ribosomal subunit profiling. (A) Exploring the synthetic growth phenotype of ΔrlmKL in the presence of the disruption of RNA helicases, including deaD, rhlE, srmB and dbpA. Each strain was serially diluted (1:10 dilutions), spotted onto LB plates and incubated at 37°C or 28°C for 11 and 22 h, respectively. The doubling time (in minutes) is shown to the right of each panel. (B) SDG profiling of ribosomal subunits in cell lysates in the presence of 0.5 mM Mg2+ for the wild-type (blue line), ΔrlmKL (red line), ΔdeaD (red line) and ΔrlmKL/ΔdeaD (red line) strains. The amount of each subunit was quantified by UV absorbance at 254 nm. The peak positions for the 30S and 50S subunits are indicated. The peak height was normalized to the 30S peak.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3351187&req=5

gkr1287-F7: Synthetic growth phenotype of ΔrlmKL and ribosomal subunit profiling. (A) Exploring the synthetic growth phenotype of ΔrlmKL in the presence of the disruption of RNA helicases, including deaD, rhlE, srmB and dbpA. Each strain was serially diluted (1:10 dilutions), spotted onto LB plates and incubated at 37°C or 28°C for 11 and 22 h, respectively. The doubling time (in minutes) is shown to the right of each panel. (B) SDG profiling of ribosomal subunits in cell lysates in the presence of 0.5 mM Mg2+ for the wild-type (blue line), ΔrlmKL (red line), ΔdeaD (red line) and ΔrlmKL/ΔdeaD (red line) strains. The amount of each subunit was quantified by UV absorbance at 254 nm. The peak positions for the 30S and 50S subunits are indicated. The peak height was normalized to the 30S peak.
Mentions: The protein product of ycbY has been reported to co-sediment with the early assembly intermediate of the 50S ribosomal subunit in E. coli (43). In addition, YcbY interacts with the DeaD and SrmB proteins, which are DEAD-box RNA helicases involved in 50S subunit formation (44–46). These results suggested that RlmKL may be involved in an early stage of the biogenesis of the 50S subunit. However, disruption of the rlmKL gene showed almost no growth phenotype even when cells were cultured at low temperature (Figure 7A), and no significant change in ribosome profile was detected (Figure 7B). Since YcbY interacts with DeaD and SrmB in E. coli (45), we explored the synthetic growth phenotype of the ΔrlmKL strain in which RNA helicases, including deaD, srmB, rhlE and dbpA, were knocked out. As shown in Figure 7A, we observed a synthetic growth phenotype with deaD, in particular at low temperature. When cultured at 37°C, the doubling time of the ΔrlmKL/ΔdeaD strain (28.4 min) was delayed by 4.5 min relative to the ΔrlmKL strain (23.9 min) and 2.9 min relative to the ΔdeaD strain (25.5 min). At 28°C, the ΔrlmKL/ΔdeaD strain exhibited a growth defect resulting in a doubling time of 99.0 min, a 50 min delay relative to the ΔrlmKL strain (49.0 min) and a 21.7 min delay relative to the ΔdeaD strain (77.3 min). This result suggested that RlmKL and the DeaD RNA helicase are cooperatively involved in efficient assembly of the 50S subunit. Therefore, the ribosome profile of the ΔrlmKL/ΔdeaD strain was analyzed by sucrose density gradient centrifugation. In the ΔdeaD strain (Figure 7B), a slight decrease in the 50S subunit compared with the wild-type strain, and an accumulation of a 40S fraction that contains an intermediate of the 50S subunit, were detected. In the ΔrlmKL/ΔdeaD strain (Figure 7B), an apparent decrease in the 50S subunit compared with the wild-type and ΔrlmKL strains and an accumulation of an intermediate 30–40S fraction were observed. These results indicated that deletion of rlmKL in the ΔdeaD background caused a considerable defect in 50S assembly.Figure 7.

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