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Determinants of the CmoB carboxymethyl transferase utilized for selective tRNA wobble modification.

Kim J, Xiao H, Koh J, Wang Y, Bonanno JB, Thomas K, Babbitt PC, Brown S, Lee YS, Almo SC - Nucleic Acids Res. (2015)

Bottom Line: We report the genetic, biochemical and structural characterization of CmoB, the enzyme that recognizes the unique metabolite carboxy-S-adenosine-L-methionine (Cx-SAM) and catalyzes a carboxymethyl transfer reaction resulting in formation of 5-oxyacetyluridine at the wobble position of tRNAs.Biochemical and genetic studies define the in vitro and in vivo selectivity for Cx-SAM as alkyl donor over the vastly more abundant SAM.Together, these studies provide mechanistic insight into the enzymatic and non-enzymatic feature of this alkyl transfer reaction which affords the broadened specificity required for tRNAs to recognize multiple synonymous codons.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461, USA.

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In vivo assay of cmo5U and mo5U formation by wild-type CmoB. Total RNA from ΔcmoB E. coli complemented with plasmid-encoded wild-type cmoB gene was extracted 3 and 6 h after induction with 0.5-mM IPTG, followed by P1 nuclease treatment and LC/MS analyses in negative ion mode. (A) mo5UMP was not detected after 3 h of induction (top), but was detected after 6 h (bottom), whereas (B) cmo5UMP was detected as early as 3 h after induction.
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Figure 4: In vivo assay of cmo5U and mo5U formation by wild-type CmoB. Total RNA from ΔcmoB E. coli complemented with plasmid-encoded wild-type cmoB gene was extracted 3 and 6 h after induction with 0.5-mM IPTG, followed by P1 nuclease treatment and LC/MS analyses in negative ion mode. (A) mo5UMP was not detected after 3 h of induction (top), but was detected after 6 h (bottom), whereas (B) cmo5UMP was detected as early as 3 h after induction.

Mentions: It was reported that total tRNA derived from cmoA-deficient mutants possessed both ho5U and mo5U, in contrast to wild-type total tRNA which possessed predominately cmo5U (13). The presence of small amounts of mo5U has also been reported previously from total RNA (23) and tRNA (24) of wild-type E. coli. When we examined P1 nuclease-treated total tRNA from wild-type E. coli by LC-MS/MS, a trace amount of mo5U was indeed detected in addition to the much more abundant cmo5U (Supplementary Figure S5). Enrichment of mo5U was observed in ΔcmoA- and ΔaroC-mutants, where aroC encodes the protein catalyzing the formation of chorismate, the immediate precursor of prephenate required for the CmoA-catalyzed transformation (Supplementary Figure S5). No significant amount of cmo5U was detected in these mutants, as these mutants are unable to synthesize Cx-SAM (12,13,25). Both mo5U and cmo5U were absent from ΔcmoB-mutant, in agreement with a previous report (13). These findings suggest that in addition to Cx-SAM-dependent cmo5U formation, CmoB catalyzes the SAM-dependent methylation of wobble ho5U in Gram-negative organisms, albeit with much lower efficiency. To investigate the in vivo generation of mo5U and cmo5U, modifications were monitored in cmoB-deficient cells complemented with a plasmid directing the inducible production of wild-type cmoB. Total RNA was extracted from cells at 3-, 6- and 24-h time points after the induction of CmoB expression, and nucleotides derived from P1 nuclease treatment were analyzed by LC-MS (Figure 4). Three hours post-induction of cmoB expression, cmo5U was detected in the sample, while no mo5U was observed. The MS peak corresponding to mo5U began to appear 6 h post-induction, confirming that CmoB is involved in the in vivo generation of both cmo5U and mo5U.


Determinants of the CmoB carboxymethyl transferase utilized for selective tRNA wobble modification.

Kim J, Xiao H, Koh J, Wang Y, Bonanno JB, Thomas K, Babbitt PC, Brown S, Lee YS, Almo SC - Nucleic Acids Res. (2015)

In vivo assay of cmo5U and mo5U formation by wild-type CmoB. Total RNA from ΔcmoB E. coli complemented with plasmid-encoded wild-type cmoB gene was extracted 3 and 6 h after induction with 0.5-mM IPTG, followed by P1 nuclease treatment and LC/MS analyses in negative ion mode. (A) mo5UMP was not detected after 3 h of induction (top), but was detected after 6 h (bottom), whereas (B) cmo5UMP was detected as early as 3 h after induction.
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Figure 4: In vivo assay of cmo5U and mo5U formation by wild-type CmoB. Total RNA from ΔcmoB E. coli complemented with plasmid-encoded wild-type cmoB gene was extracted 3 and 6 h after induction with 0.5-mM IPTG, followed by P1 nuclease treatment and LC/MS analyses in negative ion mode. (A) mo5UMP was not detected after 3 h of induction (top), but was detected after 6 h (bottom), whereas (B) cmo5UMP was detected as early as 3 h after induction.
Mentions: It was reported that total tRNA derived from cmoA-deficient mutants possessed both ho5U and mo5U, in contrast to wild-type total tRNA which possessed predominately cmo5U (13). The presence of small amounts of mo5U has also been reported previously from total RNA (23) and tRNA (24) of wild-type E. coli. When we examined P1 nuclease-treated total tRNA from wild-type E. coli by LC-MS/MS, a trace amount of mo5U was indeed detected in addition to the much more abundant cmo5U (Supplementary Figure S5). Enrichment of mo5U was observed in ΔcmoA- and ΔaroC-mutants, where aroC encodes the protein catalyzing the formation of chorismate, the immediate precursor of prephenate required for the CmoA-catalyzed transformation (Supplementary Figure S5). No significant amount of cmo5U was detected in these mutants, as these mutants are unable to synthesize Cx-SAM (12,13,25). Both mo5U and cmo5U were absent from ΔcmoB-mutant, in agreement with a previous report (13). These findings suggest that in addition to Cx-SAM-dependent cmo5U formation, CmoB catalyzes the SAM-dependent methylation of wobble ho5U in Gram-negative organisms, albeit with much lower efficiency. To investigate the in vivo generation of mo5U and cmo5U, modifications were monitored in cmoB-deficient cells complemented with a plasmid directing the inducible production of wild-type cmoB. Total RNA was extracted from cells at 3-, 6- and 24-h time points after the induction of CmoB expression, and nucleotides derived from P1 nuclease treatment were analyzed by LC-MS (Figure 4). Three hours post-induction of cmoB expression, cmo5U was detected in the sample, while no mo5U was observed. The MS peak corresponding to mo5U began to appear 6 h post-induction, confirming that CmoB is involved in the in vivo generation of both cmo5U and mo5U.

Bottom Line: We report the genetic, biochemical and structural characterization of CmoB, the enzyme that recognizes the unique metabolite carboxy-S-adenosine-L-methionine (Cx-SAM) and catalyzes a carboxymethyl transfer reaction resulting in formation of 5-oxyacetyluridine at the wobble position of tRNAs.Biochemical and genetic studies define the in vitro and in vivo selectivity for Cx-SAM as alkyl donor over the vastly more abundant SAM.Together, these studies provide mechanistic insight into the enzymatic and non-enzymatic feature of this alkyl transfer reaction which affords the broadened specificity required for tRNAs to recognize multiple synonymous codons.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461, USA.

Show MeSH