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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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Summary of the genetic organization, expression and assembly of the fragmented class Ia RNR from phage Aeh1. The map of the Aeh1 RNR operon (gDNA) shows an early phage promoter (right facing arrow) that drives expression of the operon (32). The NrdA-a, NrdA-b and NrdB polypeptides are independently translated from this message (mRNA) (8), while expression of MobE is inhibited by an RNA secondary structure (33). Our current data indicate that the NrdA-a and NrdA-b polypeptides self assemble to form the αa/αb heterodimer (active site residues depicted as spheres), while dimerization to form the (αa/αb)2 large subunit is stimulated by dATP. The ΔT mutants are defective in dimerization of the αa/αb heterodimer. Holoenzyme formation is stimulated by dATP and includes the small subunit dimer, β2. The structures of the individual polypeptides were modified from the E. coli NrdA (PBD file 4R1R) and NrdB (PDB file 1AV8) structures. In the holoenzyme model [based on Ref. (34)], the (αa/αb)2 large subunit is rotated 90° vertically relative to the free large subunit and the β2 subunit is rotated 90° horizontally relative to the free dimer.
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Figure 5: Summary of the genetic organization, expression and assembly of the fragmented class Ia RNR from phage Aeh1. The map of the Aeh1 RNR operon (gDNA) shows an early phage promoter (right facing arrow) that drives expression of the operon (32). The NrdA-a, NrdA-b and NrdB polypeptides are independently translated from this message (mRNA) (8), while expression of MobE is inhibited by an RNA secondary structure (33). Our current data indicate that the NrdA-a and NrdA-b polypeptides self assemble to form the αa/αb heterodimer (active site residues depicted as spheres), while dimerization to form the (αa/αb)2 large subunit is stimulated by dATP. The ΔT mutants are defective in dimerization of the αa/αb heterodimer. Holoenzyme formation is stimulated by dATP and includes the small subunit dimer, β2. The structures of the individual polypeptides were modified from the E. coli NrdA (PBD file 4R1R) and NrdB (PDB file 1AV8) structures. In the holoenzyme model [based on Ref. (34)], the (αa/αb)2 large subunit is rotated 90° vertically relative to the free large subunit and the β2 subunit is rotated 90° horizontally relative to the free dimer.

Mentions: Collectively, our data indicate that the unique tail sequences fused to the C- and N-termini of NrdA-a and NrdA-b are essential for enzymatic activity. We show that the tails are not required for interaction of the NrdA-a and NrdA-b fragments, as the ΔT mutants do not affect assembly of the αa/αb heterodimer. Rather, our data are consistent with a model whereby the tails function as interaction domains to promote assembly of the large subunit (αa/αb)2 dimer of heterodimers (Figures 4 and 5). Modeling of the split Aeh1 NrdA-a and NrdA-b proteins using the E. coli NrdA structure reveals that the tail sequences could lie in proximity to two α-helices verified by mutational studies of the E. coli protein as the major dimerization determinants between NrdA monomers (Figure 4) (13,14,19). The requirement for the tail sequences to assemble an (αa/αb)2 dimer of heterodimers clearly implies that the Aeh1 NrdA dimer interface was compromised by the mobE insertion. Although the molecular details of how the tails function remains to be elucidated, we note that the C-terminal tail of NrdA-a is positively charged (pI = 10.4), while the N-terminal tail of NrdA-b is negatively charged (pI = 3.8), suggesting that assembly of the (αa/αb)2 large subunit could be driven by direct charge–charge interactions between the tails, by interactions between the tails and opposing NrdA subunits or by a combination of both types of interactions. The proximity of the tails to active site residues on NrdA-a and NrdA-b is compelling and we cannot discount the possibility that the tails also function to promote assembly of the composite active site.Figure 4.


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)

Summary of the genetic organization, expression and assembly of the fragmented class Ia RNR from phage Aeh1. The map of the Aeh1 RNR operon (gDNA) shows an early phage promoter (right facing arrow) that drives expression of the operon (32). The NrdA-a, NrdA-b and NrdB polypeptides are independently translated from this message (mRNA) (8), while expression of MobE is inhibited by an RNA secondary structure (33). Our current data indicate that the NrdA-a and NrdA-b polypeptides self assemble to form the αa/αb heterodimer (active site residues depicted as spheres), while dimerization to form the (αa/αb)2 large subunit is stimulated by dATP. The ΔT mutants are defective in dimerization of the αa/αb heterodimer. Holoenzyme formation is stimulated by dATP and includes the small subunit dimer, β2. The structures of the individual polypeptides were modified from the E. coli NrdA (PBD file 4R1R) and NrdB (PDB file 1AV8) structures. In the holoenzyme model [based on Ref. (34)], the (αa/αb)2 large subunit is rotated 90° vertically relative to the free large subunit and the β2 subunit is rotated 90° horizontally relative to the free dimer.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 5: Summary of the genetic organization, expression and assembly of the fragmented class Ia RNR from phage Aeh1. The map of the Aeh1 RNR operon (gDNA) shows an early phage promoter (right facing arrow) that drives expression of the operon (32). The NrdA-a, NrdA-b and NrdB polypeptides are independently translated from this message (mRNA) (8), while expression of MobE is inhibited by an RNA secondary structure (33). Our current data indicate that the NrdA-a and NrdA-b polypeptides self assemble to form the αa/αb heterodimer (active site residues depicted as spheres), while dimerization to form the (αa/αb)2 large subunit is stimulated by dATP. The ΔT mutants are defective in dimerization of the αa/αb heterodimer. Holoenzyme formation is stimulated by dATP and includes the small subunit dimer, β2. The structures of the individual polypeptides were modified from the E. coli NrdA (PBD file 4R1R) and NrdB (PDB file 1AV8) structures. In the holoenzyme model [based on Ref. (34)], the (αa/αb)2 large subunit is rotated 90° vertically relative to the free large subunit and the β2 subunit is rotated 90° horizontally relative to the free dimer.
Mentions: Collectively, our data indicate that the unique tail sequences fused to the C- and N-termini of NrdA-a and NrdA-b are essential for enzymatic activity. We show that the tails are not required for interaction of the NrdA-a and NrdA-b fragments, as the ΔT mutants do not affect assembly of the αa/αb heterodimer. Rather, our data are consistent with a model whereby the tails function as interaction domains to promote assembly of the large subunit (αa/αb)2 dimer of heterodimers (Figures 4 and 5). Modeling of the split Aeh1 NrdA-a and NrdA-b proteins using the E. coli NrdA structure reveals that the tail sequences could lie in proximity to two α-helices verified by mutational studies of the E. coli protein as the major dimerization determinants between NrdA monomers (Figure 4) (13,14,19). The requirement for the tail sequences to assemble an (αa/αb)2 dimer of heterodimers clearly implies that the Aeh1 NrdA dimer interface was compromised by the mobE insertion. Although the molecular details of how the tails function remains to be elucidated, we note that the C-terminal tail of NrdA-a is positively charged (pI = 10.4), while the N-terminal tail of NrdA-b is negatively charged (pI = 3.8), suggesting that assembly of the (αa/αb)2 large subunit could be driven by direct charge–charge interactions between the tails, by interactions between the tails and opposing NrdA subunits or by a combination of both types of interactions. The proximity of the tails to active site residues on NrdA-a and NrdA-b is compelling and we cannot discount the possibility that the tails also function to promote assembly of the composite active site.Figure 4.

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