Limits...
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.

Show MeSH

Related in: MedlinePlus

Schematic depiction of a catalytic mechanism for the cooperative modifications of Helix 74. After transcription of domain V in the 23S rRNA, the C-terminal RlmK domain of RlmKL unwinds Helix 74 upon recognizing Helix 80 and Helix 74, and then methylates G2069. The single-stranded 3′ side of Helix 74 is then recognized by the N-terminal RlmL domain, and G2445 is methylated. Finally, Helix 74 reforms a duplex, and domain V is restructured.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3351187&req=5

gkr1287-F8: Schematic depiction of a catalytic mechanism for the cooperative modifications of Helix 74. After transcription of domain V in the 23S rRNA, the C-terminal RlmK domain of RlmKL unwinds Helix 74 upon recognizing Helix 80 and Helix 74, and then methylates G2069. The single-stranded 3′ side of Helix 74 is then recognized by the N-terminal RlmL domain, and G2445 is methylated. Finally, Helix 74 reforms a duplex, and domain V is restructured.

Mentions: Based on this study, we propose a catalytic mechanism for the cooperative modifications of Helix 74 (Figure 8). Our biochemical data has shown that RlmK recognizes Helix 80, the conserved bases in the P-loop, 12 nt ss region and Helix74, whereas RlmL recognizes critical residues in the 12 nt ss region and the 3′ strand of Helix 74 (Figure 6D). As shown in the crystal structure of the 50S subunit (Figure 6E), the residues required for m2G2445 formation are interrupted by the residues essential for m7G2069 formation. Thus, both domains of RlmKL cannot bind Helix 74 at the same time in the folded structure of the 23S rRNA. In addition, RlmL(NTD) preferentially recognizes and methylates the single-stranded substrate rather than the duplex substrate, suggesting that Helix 74 should be unwound to serve as the preferred substrate for RlmL(NTD) during dimethyl-formation. In fact, we could directly observe an unwinding activity of RlmKL with the duplex substrate formed by transcripts 8 and 9 (Figure 5C), strongly supporting the proposed mechanism of cooperative methylation by RlmKL. As discussed above, RlmK has an EEHEE domain that may recognize the 5′ residues neighboring G2069 in Helix 74. Since several residues in the 5′ strand of Helix 74 are essential for m7G2069 formation, the EEHEE domain may recognize these bases and unwind Helix 74, releasing the 3′ ss for RlmL(NTD) recognition.Figure 8.


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)

Schematic depiction of a catalytic mechanism for the cooperative modifications of Helix 74. After transcription of domain V in the 23S rRNA, the C-terminal RlmK domain of RlmKL unwinds Helix 74 upon recognizing Helix 80 and Helix 74, and then methylates G2069. The single-stranded 3′ side of Helix 74 is then recognized by the N-terminal RlmL domain, and G2445 is methylated. Finally, Helix 74 reforms a duplex, and domain V is restructured.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkr1287-F8: Schematic depiction of a catalytic mechanism for the cooperative modifications of Helix 74. After transcription of domain V in the 23S rRNA, the C-terminal RlmK domain of RlmKL unwinds Helix 74 upon recognizing Helix 80 and Helix 74, and then methylates G2069. The single-stranded 3′ side of Helix 74 is then recognized by the N-terminal RlmL domain, and G2445 is methylated. Finally, Helix 74 reforms a duplex, and domain V is restructured.
Mentions: Based on this study, we propose a catalytic mechanism for the cooperative modifications of Helix 74 (Figure 8). Our biochemical data has shown that RlmK recognizes Helix 80, the conserved bases in the P-loop, 12 nt ss region and Helix74, whereas RlmL recognizes critical residues in the 12 nt ss region and the 3′ strand of Helix 74 (Figure 6D). As shown in the crystal structure of the 50S subunit (Figure 6E), the residues required for m2G2445 formation are interrupted by the residues essential for m7G2069 formation. Thus, both domains of RlmKL cannot bind Helix 74 at the same time in the folded structure of the 23S rRNA. In addition, RlmL(NTD) preferentially recognizes and methylates the single-stranded substrate rather than the duplex substrate, suggesting that Helix 74 should be unwound to serve as the preferred substrate for RlmL(NTD) during dimethyl-formation. In fact, we could directly observe an unwinding activity of RlmKL with the duplex substrate formed by transcripts 8 and 9 (Figure 5C), strongly supporting the proposed mechanism of cooperative methylation by RlmKL. As discussed above, RlmK has an EEHEE domain that may recognize the 5′ residues neighboring G2069 in Helix 74. Since several residues in the 5′ strand of Helix 74 are essential for m7G2069 formation, the EEHEE domain may recognize these bases and unwind Helix 74, releasing the 3′ ss for RlmL(NTD) recognition.Figure 8.

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