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Assembly of a fragmented ribonucleotide reductase by protein interaction domains derived from a mobile genetic element.

Crona M, Moffatt C, Friedrich NC, Hofer A, Sjöberg BM, Edgell DR - Nucleic Acids Res. (2010)

Bottom Line: Ribonucleotide reductase (RNR) is a critical enzyme of nucleotide metabolism, synthesizing precursors for DNA replication and repair.In prokaryotic genomes, RNR genes are commonly targeted by mobile genetic elements, including free standing and intron-encoded homing endonucleases and inteins.Our data are consistent with the tails functioning as protein interaction domains to assemble the tetrameric (NrdA-a/NrdA-b)(2) large subunit necessary for a functional RNR holoenzyme.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Functional Genomics, Stockholm University, S-10691 Stockholm, Sweden.

ABSTRACT
Ribonucleotide reductase (RNR) is a critical enzyme of nucleotide metabolism, synthesizing precursors for DNA replication and repair. In prokaryotic genomes, RNR genes are commonly targeted by mobile genetic elements, including free standing and intron-encoded homing endonucleases and inteins. Here, we describe a unique molecular solution to assemble a functional product from the RNR large subunit gene, nrdA that has been fragmented into two smaller genes by the insertion of mobE, a mobile endonuclease. We show that unique sequences that originated during the mobE insertion and that are present as C- and N-terminal tails on the split NrdA-a and NrdA-b polypeptides, are absolutely essential for enzymatic activity. Our data are consistent with the tails functioning as protein interaction domains to assemble the tetrameric (NrdA-a/NrdA-b)(2) large subunit necessary for a functional RNR holoenzyme. The tails represent a solution distinct from RNA and protein splicing or programmed DNA rearrangements to restore function from a fragmented coding region and may represent a general mechanism to neutralize fragmentation of essential genes by mobile genetic elements.

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The mutant αaΔT/αbΔT heterodimer is defective in dATP-mediated dimerization. Shown are representative GEMMA analyses of the αa/αb or αaΔT/αbΔT heterodimers with no added nucleotide (traces A and B), or with 50 mM dATP (traces C and D). The concentration of the αa/αb heterodimer was 0.01 mg/ml (0.11 µM) and the concentration of the αaΔT/αbΔT heterodimer was 0.04 mg/ml (0.47 µM). For each condition, the composition and predicted sizes (in kDa) of the species are indicated. The baseline has been shifted by 1000 intensity counts for each trace.
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Figure 2: The mutant αaΔT/αbΔT heterodimer is defective in dATP-mediated dimerization. Shown are representative GEMMA analyses of the αa/αb or αaΔT/αbΔT heterodimers with no added nucleotide (traces A and B), or with 50 mM dATP (traces C and D). The concentration of the αa/αb heterodimer was 0.01 mg/ml (0.11 µM) and the concentration of the αaΔT/αbΔT heterodimer was 0.04 mg/ml (0.47 µM). For each condition, the composition and predicted sizes (in kDa) of the species are indicated. The baseline has been shifted by 1000 intensity counts for each trace.

Mentions: We hypothesized that one mechanism by which the tails are required for RNR activity would be to promote or stabilize interactions between NrdA-a and NrdA-b. To test this hypothesis, we examined the oligomeric status of the split wild-type and mutant ΔT NrdA proteins by gas-phase electrophoretic mobility macromolecular analysis (GEMMA) (17), a technique that measures the diameter of protein complexes in the gas phase at low protein concentrations. In the absence of exogenously added nucleotides, the sizes of the major species observed are consistent with heterodimers of the wild type (αa/αb) and tail mutant proteins (αaΔT/αbΔT), respectively (Figure 2A and B; Table 1). Furthermore, we analyzed the wild-type and ΔT NrdA proteins by analytical ultracentrifugation at higher protein concentrations (∼0.2–0.6 mg/ml) in the absence of nucleotides and found that the sizes were consistent with the proteins existing as heterodimeric αa/αb and αaΔT/αbΔT species (Table 1 and Supplementary Figure S2). Velocity ultracentrifugation, which in contrast to equilibrium ultracentrifugation can detect changes in the shape of proteins, showed that the heterodimeric wild-type and ΔT complexes possessed different sedimentation coefficients (6.1S versus 4.5S) that are too large to be accounted for by differences in the predicted molecular weights of the heterodimers (Supplementary Figure S2). These results indicated that deletion of the tails affects the shape of the αaΔT/αbΔT complex relative to the αa/αb complex. Potential differences in shape were also evident upon gel-filtration chromatography, as the elution profiles of the αa/αb and αaΔT/αbΔT heterodimers were shifted relative to each other (Supplementary Figure S2). Collectively, these data indicate that the tails are not required for interaction of αa with αb to form a heterodimer and that deletion of the tails affects the shape of the heterodimer.Figure 2.


Assembly of a fragmented ribonucleotide reductase by protein interaction domains derived from a mobile genetic element.

Crona M, Moffatt C, Friedrich NC, Hofer A, Sjöberg BM, Edgell DR - Nucleic Acids Res. (2010)

The mutant αaΔT/αbΔT heterodimer is defective in dATP-mediated dimerization. Shown are representative GEMMA analyses of the αa/αb or αaΔT/αbΔT heterodimers with no added nucleotide (traces A and B), or with 50 mM dATP (traces C and D). The concentration of the αa/αb heterodimer was 0.01 mg/ml (0.11 µM) and the concentration of the αaΔT/αbΔT heterodimer was 0.04 mg/ml (0.47 µM). For each condition, the composition and predicted sizes (in kDa) of the species are indicated. The baseline has been shifted by 1000 intensity counts for each trace.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3045599&req=5

Figure 2: The mutant αaΔT/αbΔT heterodimer is defective in dATP-mediated dimerization. Shown are representative GEMMA analyses of the αa/αb or αaΔT/αbΔT heterodimers with no added nucleotide (traces A and B), or with 50 mM dATP (traces C and D). The concentration of the αa/αb heterodimer was 0.01 mg/ml (0.11 µM) and the concentration of the αaΔT/αbΔT heterodimer was 0.04 mg/ml (0.47 µM). For each condition, the composition and predicted sizes (in kDa) of the species are indicated. The baseline has been shifted by 1000 intensity counts for each trace.
Mentions: We hypothesized that one mechanism by which the tails are required for RNR activity would be to promote or stabilize interactions between NrdA-a and NrdA-b. To test this hypothesis, we examined the oligomeric status of the split wild-type and mutant ΔT NrdA proteins by gas-phase electrophoretic mobility macromolecular analysis (GEMMA) (17), a technique that measures the diameter of protein complexes in the gas phase at low protein concentrations. In the absence of exogenously added nucleotides, the sizes of the major species observed are consistent with heterodimers of the wild type (αa/αb) and tail mutant proteins (αaΔT/αbΔT), respectively (Figure 2A and B; Table 1). Furthermore, we analyzed the wild-type and ΔT NrdA proteins by analytical ultracentrifugation at higher protein concentrations (∼0.2–0.6 mg/ml) in the absence of nucleotides and found that the sizes were consistent with the proteins existing as heterodimeric αa/αb and αaΔT/αbΔT species (Table 1 and Supplementary Figure S2). Velocity ultracentrifugation, which in contrast to equilibrium ultracentrifugation can detect changes in the shape of proteins, showed that the heterodimeric wild-type and ΔT complexes possessed different sedimentation coefficients (6.1S versus 4.5S) that are too large to be accounted for by differences in the predicted molecular weights of the heterodimers (Supplementary Figure S2). These results indicated that deletion of the tails affects the shape of the αaΔT/αbΔT complex relative to the αa/αb complex. Potential differences in shape were also evident upon gel-filtration chromatography, as the elution profiles of the αa/αb and αaΔT/αbΔT heterodimers were shifted relative to each other (Supplementary Figure S2). Collectively, these data indicate that the tails are not required for interaction of αa with αb to form a heterodimer and that deletion of the tails affects the shape of the heterodimer.Figure 2.

Bottom Line: Ribonucleotide reductase (RNR) is a critical enzyme of nucleotide metabolism, synthesizing precursors for DNA replication and repair.In prokaryotic genomes, RNR genes are commonly targeted by mobile genetic elements, including free standing and intron-encoded homing endonucleases and inteins.Our data are consistent with the tails functioning as protein interaction domains to assemble the tetrameric (NrdA-a/NrdA-b)(2) large subunit necessary for a functional RNR holoenzyme.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Functional Genomics, Stockholm University, S-10691 Stockholm, Sweden.

ABSTRACT
Ribonucleotide reductase (RNR) is a critical enzyme of nucleotide metabolism, synthesizing precursors for DNA replication and repair. In prokaryotic genomes, RNR genes are commonly targeted by mobile genetic elements, including free standing and intron-encoded homing endonucleases and inteins. Here, we describe a unique molecular solution to assemble a functional product from the RNR large subunit gene, nrdA that has been fragmented into two smaller genes by the insertion of mobE, a mobile endonuclease. We show that unique sequences that originated during the mobE insertion and that are present as C- and N-terminal tails on the split NrdA-a and NrdA-b polypeptides, are absolutely essential for enzymatic activity. Our data are consistent with the tails functioning as protein interaction domains to assemble the tetrameric (NrdA-a/NrdA-b)(2) large subunit necessary for a functional RNR holoenzyme. The tails represent a solution distinct from RNA and protein splicing or programmed DNA rearrangements to restore function from a fragmented coding region and may represent a general mechanism to neutralize fragmentation of essential genes by mobile genetic elements.

Show MeSH
Related in: MedlinePlus