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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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Related in: MedlinePlus

The mobE insertion fragments the active site of the class I RNR of phage Aeh1. (A) Schematic of the Aeh1 nrdA-a, mobE and nrdA-b genes, with the GC content over a 100 nucleotide-sliding window plotted above. The average GC content over the operon (42%) is indicated by a dashed line. Note that the mobE and nrdA-b genes overlap. Shown below is an amino acid alignment of the Aeh1 NrdA-a and NrdA-b and related NrdA proteins, with the mobE insertion indicated by a right facing arrow (not to scale). Active site residues of E. coli NrdA are indicated by solid triangles and conserved or identical amino acids are shaded gray or black with white lettering, respectively. The Aeh1 NrdA-a and NrdA-b tails are highlighted by rectangles. (B) Left, the E. coli NrdA monomer, colored to indicate the split between the Aeh1 NrdA-a (yellow) and NrdA-b (green) proteins. Active site residues are depicted as red spheres. Right, model of the NrdA-a and NrdA-b fragments based on the E. coli NrdA structure, with active site residues indicated by red spheres. The NrdA-b fragment is rotated relative to its position in the monomer to highlight the active site residues. (C) Subunit composition of the large subunit or holoenzyme of the class Ia RNR for the prototypical E. coli enzyme or the phage Aeh1 enzyme.
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Figure 1: The mobE insertion fragments the active site of the class I RNR of phage Aeh1. (A) Schematic of the Aeh1 nrdA-a, mobE and nrdA-b genes, with the GC content over a 100 nucleotide-sliding window plotted above. The average GC content over the operon (42%) is indicated by a dashed line. Note that the mobE and nrdA-b genes overlap. Shown below is an amino acid alignment of the Aeh1 NrdA-a and NrdA-b and related NrdA proteins, with the mobE insertion indicated by a right facing arrow (not to scale). Active site residues of E. coli NrdA are indicated by solid triangles and conserved or identical amino acids are shaded gray or black with white lettering, respectively. The Aeh1 NrdA-a and NrdA-b tails are highlighted by rectangles. (B) Left, the E. coli NrdA monomer, colored to indicate the split between the Aeh1 NrdA-a (yellow) and NrdA-b (green) proteins. Active site residues are depicted as red spheres. Right, model of the NrdA-a and NrdA-b fragments based on the E. coli NrdA structure, with active site residues indicated by red spheres. The NrdA-b fragment is rotated relative to its position in the monomer to highlight the active site residues. (C) Subunit composition of the large subunit or holoenzyme of the class Ia RNR for the prototypical E. coli enzyme or the phage Aeh1 enzyme.

Mentions: Here, we examine how function is restored when a ribonucleotide reductase (RNR) gene has been fragmented by the insertion of a mobile genetic element such that RNR active site residues are partitioned between two genes. In phage Aeh1 that infects Aeromonas hydrophila, the nrdA gene for the large subunit of aerobic RNR has been fragmented into two smaller genes, nrdA-a and nrdA-b, by the transposition of the homing endonuclease mobE (Figure 1A) (8). RNRs are functionally critical enzymes that catalyze the synthesis of deoxyribonucleotides used for DNA replication and repair (9) and are common targets of homing endonucleases (10–12). Class Ia RNRs require the presence of the NrdB component encoded by the nrdB gene and in bacteria and phage the active holoenzyme is generally a tetramer composed of a dimer of the large subunit NrdA protein (α2) and a dimer of the small subunit NrdB protein (β2; see Figure 1 for nomenclature). The mobE insertion splits the Aeh1 nrdA gene at a position that in the Escherichia coli NrdA structure lies between two adjacent β-strands in the RNR specific 10-stranded α/β-barrel that constitutes the active site (Figure 1B). Three active site residues, Cys-219 and Cys-431 in NrdA-a and Cys-31 in NrdA-b, are located in separate polypeptides, while the homologous residues in E. coli NrdA (Cys-225, Cys-439 and Cys-462) are present in the same polypeptide (13). An essential part of the reaction mechanism involves a transient disulfide bond formed between Cys-225 and Cys-462 in E. coli NrdA (9,14). The corresponding residues in Aeh1 are Cys-219 located in NrdA-a and Cys-31 located in NrdA-b. Moreover, in 1200 nrdA sequences from viruses as well as cellular organisms, none are split into multiple coding regions (15), suggesting strong evolutionary pressure to retain NrdA as a continuous polypeptide. Recent metagenomic data has, however, revealed the presence of nrdA genes fragmented by the insertion of inteins that presumably undergo trans-splicing to restore NrdA function (7). Remarkably, the Aeh1 holoenzyme is fully functional with specific activity equivalent to other characterized class Ia RNRs, but with an unusual (NrdA-a/NrdA-b)2NrdB2 or (αa/αb)2/β2, subunit composition (Figure 1C) (8). Thus, a composite active site and functional holoenzyme is assembled from residues on each polypeptide.Figure 1.


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 mobE insertion fragments the active site of the class I RNR of phage Aeh1. (A) Schematic of the Aeh1 nrdA-a, mobE and nrdA-b genes, with the GC content over a 100 nucleotide-sliding window plotted above. The average GC content over the operon (42%) is indicated by a dashed line. Note that the mobE and nrdA-b genes overlap. Shown below is an amino acid alignment of the Aeh1 NrdA-a and NrdA-b and related NrdA proteins, with the mobE insertion indicated by a right facing arrow (not to scale). Active site residues of E. coli NrdA are indicated by solid triangles and conserved or identical amino acids are shaded gray or black with white lettering, respectively. The Aeh1 NrdA-a and NrdA-b tails are highlighted by rectangles. (B) Left, the E. coli NrdA monomer, colored to indicate the split between the Aeh1 NrdA-a (yellow) and NrdA-b (green) proteins. Active site residues are depicted as red spheres. Right, model of the NrdA-a and NrdA-b fragments based on the E. coli NrdA structure, with active site residues indicated by red spheres. The NrdA-b fragment is rotated relative to its position in the monomer to highlight the active site residues. (C) Subunit composition of the large subunit or holoenzyme of the class Ia RNR for the prototypical E. coli enzyme or the phage Aeh1 enzyme.
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Related In: Results  -  Collection

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Figure 1: The mobE insertion fragments the active site of the class I RNR of phage Aeh1. (A) Schematic of the Aeh1 nrdA-a, mobE and nrdA-b genes, with the GC content over a 100 nucleotide-sliding window plotted above. The average GC content over the operon (42%) is indicated by a dashed line. Note that the mobE and nrdA-b genes overlap. Shown below is an amino acid alignment of the Aeh1 NrdA-a and NrdA-b and related NrdA proteins, with the mobE insertion indicated by a right facing arrow (not to scale). Active site residues of E. coli NrdA are indicated by solid triangles and conserved or identical amino acids are shaded gray or black with white lettering, respectively. The Aeh1 NrdA-a and NrdA-b tails are highlighted by rectangles. (B) Left, the E. coli NrdA monomer, colored to indicate the split between the Aeh1 NrdA-a (yellow) and NrdA-b (green) proteins. Active site residues are depicted as red spheres. Right, model of the NrdA-a and NrdA-b fragments based on the E. coli NrdA structure, with active site residues indicated by red spheres. The NrdA-b fragment is rotated relative to its position in the monomer to highlight the active site residues. (C) Subunit composition of the large subunit or holoenzyme of the class Ia RNR for the prototypical E. coli enzyme or the phage Aeh1 enzyme.
Mentions: Here, we examine how function is restored when a ribonucleotide reductase (RNR) gene has been fragmented by the insertion of a mobile genetic element such that RNR active site residues are partitioned between two genes. In phage Aeh1 that infects Aeromonas hydrophila, the nrdA gene for the large subunit of aerobic RNR has been fragmented into two smaller genes, nrdA-a and nrdA-b, by the transposition of the homing endonuclease mobE (Figure 1A) (8). RNRs are functionally critical enzymes that catalyze the synthesis of deoxyribonucleotides used for DNA replication and repair (9) and are common targets of homing endonucleases (10–12). Class Ia RNRs require the presence of the NrdB component encoded by the nrdB gene and in bacteria and phage the active holoenzyme is generally a tetramer composed of a dimer of the large subunit NrdA protein (α2) and a dimer of the small subunit NrdB protein (β2; see Figure 1 for nomenclature). The mobE insertion splits the Aeh1 nrdA gene at a position that in the Escherichia coli NrdA structure lies between two adjacent β-strands in the RNR specific 10-stranded α/β-barrel that constitutes the active site (Figure 1B). Three active site residues, Cys-219 and Cys-431 in NrdA-a and Cys-31 in NrdA-b, are located in separate polypeptides, while the homologous residues in E. coli NrdA (Cys-225, Cys-439 and Cys-462) are present in the same polypeptide (13). An essential part of the reaction mechanism involves a transient disulfide bond formed between Cys-225 and Cys-462 in E. coli NrdA (9,14). The corresponding residues in Aeh1 are Cys-219 located in NrdA-a and Cys-31 located in NrdA-b. Moreover, in 1200 nrdA sequences from viruses as well as cellular organisms, none are split into multiple coding regions (15), suggesting strong evolutionary pressure to retain NrdA as a continuous polypeptide. Recent metagenomic data has, however, revealed the presence of nrdA genes fragmented by the insertion of inteins that presumably undergo trans-splicing to restore NrdA function (7). Remarkably, the Aeh1 holoenzyme is fully functional with specific activity equivalent to other characterized class Ia RNRs, but with an unusual (NrdA-a/NrdA-b)2NrdB2 or (αa/αb)2/β2, subunit composition (Figure 1C) (8). Thus, a composite active site and functional holoenzyme is assembled from residues on each polypeptide.Figure 1.

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