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PrrC-anticodon nuclease: functional organization of a prototypical bacterial restriction RNase.

Blanga-Kanfi S, Amitsur M, Azem A, Kaufmann G - Nucleic Acids Res. (2006)

Bottom Line: The tRNA(Lys) anticodon nuclease PrrC is associated in latent form with the type Ic DNA restriction endonuclease EcoprrI and activated by a phage T4-encoded inhibitor of EcoprrI.Microbiol., 50, 129-143].The differential conservation and consistent genetic linkage of the PrrC proteins with EcoprrI homologs portray them as a family of restriction RNases of diverse substrate specificities that are mobilized when an associated DNA restriction nuclease is compromised.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Tel Aviv University, Ramat Aviv 69978, Israel.

ABSTRACT
The tRNA(Lys) anticodon nuclease PrrC is associated in latent form with the type Ic DNA restriction endonuclease EcoprrI and activated by a phage T4-encoded inhibitor of EcoprrI. The activation also requires the hydrolysis of GTP and presence of dTTP and is inhibited by ATP. The N-proximal NTPase domain of PrrC has been implicated in relaying the activating signal to a C-proximal anticodon nuclease site by interacting with the requisite nucleotide cofactors [Amitsur et al. (2003) Mol. Microbiol., 50, 129-143]. Means described here to bypass PrrC's self-limiting translation and thermal instability allowed purifying an active mutant form of the protein, demonstrating its oligomeric structure and confirming its anticipated interactions with the nucleotide cofactors of the activation reaction. Mutagenesis and chemical rescue data shown implicate the C-proximal Arg320, Glu324 and, possibly, His356 in anticodon nuclease catalysis. This triad exists in all the known PrrC homologs but only some of them feature residues needed for tRNA(Lys) recognition by the Escherichia coli prototype. The differential conservation and consistent genetic linkage of the PrrC proteins with EcoprrI homologs portray them as a family of restriction RNases of diverse substrate specificities that are mobilized when an associated DNA restriction nuclease is compromised.

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Protein levels and ACNase activities of wild-type and D222E alleles of PrrC. (A) In vivo levels of protein and ACNase cleavage products of the two PrrC forms. The expression of the two forms was induced by IPTG and the cellular levels of their protein and ACNase cleavage products were then determined as detailed in Materials and Methods. (B) In vitro ACNase activity of the indicated PrrC forms was determined as detailed in Materials and Methods. (C) Decay rates of the in vitro ACNase activity of wild-type and D222E forms of PrrC. The PrrC-D222E fraction was assayed as such or diluted 20-fold in an isogenic extract lacking PrrC. The dilution equalized the protein levels of the mutant and wild-type forms. Aliquots were pre-incubated at 37°C in the presence of 2 M TMAO and assayed for remaining ACNase activity at 10°C and 0.5 M TMAO under standard assay conditions. D222E 1:20 indicates D222E diluted 20-fold. (D) Levels of PrrC-D222E protein in the indicated E.coli strains.
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fig2: Protein levels and ACNase activities of wild-type and D222E alleles of PrrC. (A) In vivo levels of protein and ACNase cleavage products of the two PrrC forms. The expression of the two forms was induced by IPTG and the cellular levels of their protein and ACNase cleavage products were then determined as detailed in Materials and Methods. (B) In vitro ACNase activity of the indicated PrrC forms was determined as detailed in Materials and Methods. (C) Decay rates of the in vitro ACNase activity of wild-type and D222E forms of PrrC. The PrrC-D222E fraction was assayed as such or diluted 20-fold in an isogenic extract lacking PrrC. The dilution equalized the protein levels of the mutant and wild-type forms. Aliquots were pre-incubated at 37°C in the presence of 2 M TMAO and assayed for remaining ACNase activity at 10°C and 0.5 M TMAO under standard assay conditions. D222E 1:20 indicates D222E diluted 20-fold. (D) Levels of PrrC-D222E protein in the indicated E.coli strains.

Mentions: Expression of PrrC inactivates essential tRNAs and inhibits translation, including PrrC's. However, PrrC mutants impaired in ACNase activity accumulate to higher levels, inversely proportional to the residual activity (23). This observation suggested that leaky PrrC mutants would facilitate the isolation of active core ACNase forms. One of them, PrrC-D222E, was chosen for further work due to its relatively high in vitro stability. Cells expressing PrrC-D222E featured lower in vivo ACNase activity than wild-type PrrC (23) (Figure 2A). Yet, the cell-free extract of the PrrC-D222E mutant was as active as the wild-type counterpart (Figure 2B). This difference was due to the higher in vitro stability of the mutant activity and, in turn, the higher level of the mutant protein. Namely, in the original cell extract the mutant activity decayed slower than the wild-type but similar decay rates were observed when the concentrations of the two PrrC forms in the cell extract were equalized (Figure 2C). The PrrC protein yield could be further increased by expression in the Rosetta strain (Figure 2D), thus compensating for the abundant rare codons in prrC. However, co-expressing T4 Pnk and Rli1 to counteract ACNase cleavage increased the yield of wild-type PrrC only (data not shown). Thus, the relatively weak activity of PrrC-D222E did not limit its translation.


PrrC-anticodon nuclease: functional organization of a prototypical bacterial restriction RNase.

Blanga-Kanfi S, Amitsur M, Azem A, Kaufmann G - Nucleic Acids Res. (2006)

Protein levels and ACNase activities of wild-type and D222E alleles of PrrC. (A) In vivo levels of protein and ACNase cleavage products of the two PrrC forms. The expression of the two forms was induced by IPTG and the cellular levels of their protein and ACNase cleavage products were then determined as detailed in Materials and Methods. (B) In vitro ACNase activity of the indicated PrrC forms was determined as detailed in Materials and Methods. (C) Decay rates of the in vitro ACNase activity of wild-type and D222E forms of PrrC. The PrrC-D222E fraction was assayed as such or diluted 20-fold in an isogenic extract lacking PrrC. The dilution equalized the protein levels of the mutant and wild-type forms. Aliquots were pre-incubated at 37°C in the presence of 2 M TMAO and assayed for remaining ACNase activity at 10°C and 0.5 M TMAO under standard assay conditions. D222E 1:20 indicates D222E diluted 20-fold. (D) Levels of PrrC-D222E protein in the indicated E.coli strains.
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Related In: Results  -  Collection

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fig2: Protein levels and ACNase activities of wild-type and D222E alleles of PrrC. (A) In vivo levels of protein and ACNase cleavage products of the two PrrC forms. The expression of the two forms was induced by IPTG and the cellular levels of their protein and ACNase cleavage products were then determined as detailed in Materials and Methods. (B) In vitro ACNase activity of the indicated PrrC forms was determined as detailed in Materials and Methods. (C) Decay rates of the in vitro ACNase activity of wild-type and D222E forms of PrrC. The PrrC-D222E fraction was assayed as such or diluted 20-fold in an isogenic extract lacking PrrC. The dilution equalized the protein levels of the mutant and wild-type forms. Aliquots were pre-incubated at 37°C in the presence of 2 M TMAO and assayed for remaining ACNase activity at 10°C and 0.5 M TMAO under standard assay conditions. D222E 1:20 indicates D222E diluted 20-fold. (D) Levels of PrrC-D222E protein in the indicated E.coli strains.
Mentions: Expression of PrrC inactivates essential tRNAs and inhibits translation, including PrrC's. However, PrrC mutants impaired in ACNase activity accumulate to higher levels, inversely proportional to the residual activity (23). This observation suggested that leaky PrrC mutants would facilitate the isolation of active core ACNase forms. One of them, PrrC-D222E, was chosen for further work due to its relatively high in vitro stability. Cells expressing PrrC-D222E featured lower in vivo ACNase activity than wild-type PrrC (23) (Figure 2A). Yet, the cell-free extract of the PrrC-D222E mutant was as active as the wild-type counterpart (Figure 2B). This difference was due to the higher in vitro stability of the mutant activity and, in turn, the higher level of the mutant protein. Namely, in the original cell extract the mutant activity decayed slower than the wild-type but similar decay rates were observed when the concentrations of the two PrrC forms in the cell extract were equalized (Figure 2C). The PrrC protein yield could be further increased by expression in the Rosetta strain (Figure 2D), thus compensating for the abundant rare codons in prrC. However, co-expressing T4 Pnk and Rli1 to counteract ACNase cleavage increased the yield of wild-type PrrC only (data not shown). Thus, the relatively weak activity of PrrC-D222E did not limit its translation.

Bottom Line: The tRNA(Lys) anticodon nuclease PrrC is associated in latent form with the type Ic DNA restriction endonuclease EcoprrI and activated by a phage T4-encoded inhibitor of EcoprrI.Microbiol., 50, 129-143].The differential conservation and consistent genetic linkage of the PrrC proteins with EcoprrI homologs portray them as a family of restriction RNases of diverse substrate specificities that are mobilized when an associated DNA restriction nuclease is compromised.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Tel Aviv University, Ramat Aviv 69978, Israel.

ABSTRACT
The tRNA(Lys) anticodon nuclease PrrC is associated in latent form with the type Ic DNA restriction endonuclease EcoprrI and activated by a phage T4-encoded inhibitor of EcoprrI. The activation also requires the hydrolysis of GTP and presence of dTTP and is inhibited by ATP. The N-proximal NTPase domain of PrrC has been implicated in relaying the activating signal to a C-proximal anticodon nuclease site by interacting with the requisite nucleotide cofactors [Amitsur et al. (2003) Mol. Microbiol., 50, 129-143]. Means described here to bypass PrrC's self-limiting translation and thermal instability allowed purifying an active mutant form of the protein, demonstrating its oligomeric structure and confirming its anticipated interactions with the nucleotide cofactors of the activation reaction. Mutagenesis and chemical rescue data shown implicate the C-proximal Arg320, Glu324 and, possibly, His356 in anticodon nuclease catalysis. This triad exists in all the known PrrC homologs but only some of them feature residues needed for tRNA(Lys) recognition by the Escherichia coli prototype. The differential conservation and consistent genetic linkage of the PrrC proteins with EcoprrI homologs portray them as a family of restriction RNases of diverse substrate specificities that are mobilized when an associated DNA restriction nuclease is compromised.

Show MeSH
Related in: MedlinePlus