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YtqI from Bacillus subtilis has both oligoribonuclease and pAp-phosphatase activity.

Mechold U, Fang G, Ngo S, Ogryzko V, Danchin A - Nucleic Acids Res. (2007)

Bottom Line: Firmicutes including Bacillus subtilis do not have an Oligoribonuclease (Orn) homologous protein and it is not yet understood which proteins accomplish the equivalent function in these organisms.An ytqI mutant in B. subtilis shows impairment of growth in the absence of cysteine, a phenotype resembling that of a cysQ mutant in E. coli.Phylogenetic distribution of YtqI, Orn and CysQ supports bifunctionality of YtqI.

View Article: PubMed Central - PubMed

Affiliation: Institut Pasteur, URA 2171, Unité de Génétique des Génomes Bactériens, 75724 Paris Cedex 15, France. umechold@pasteur.fr

ABSTRACT
Oligoribonuclease is the only RNase in Escherichia coli that is able to degrade RNA oligonucleotides five residues and shorter in length. Firmicutes including Bacillus subtilis do not have an Oligoribonuclease (Orn) homologous protein and it is not yet understood which proteins accomplish the equivalent function in these organisms. We had previously identified oligoribonucleases Orn from E. coli and its human homolog Sfn in a screen for proteins that are regulated by 3'-phosphoadenosine 5'-phosphate (pAp). Here, we identify YtqI as a potential functional analog of Orn through its interaction with pAp. YtqI degrades RNA oligonucleotides in vitro with preference for 3-mers. In addition, YtqI has pAp-phosphatase activity in vitro. In agreement with these data, YtqI is able to complement both orn and cysQ mutants in E. coli. An ytqI mutant in B. subtilis shows impairment of growth in the absence of cysteine, a phenotype resembling that of a cysQ mutant in E. coli. Phylogenetic distribution of YtqI, Orn and CysQ supports bifunctionality of YtqI.

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Comparison of YtqI and Orn-catalyzed degradation of nanoRNA 5-mers. Shown are the separation of reaction products on 22% PAA gels (upper panel) and the corresponding quantification (lower panel). Reactions contained 12 μg YtqI (A) or 0.14 μg Orn (B) and 1.5 μM or 2.7 μM RNA 5-mer (5′Cy5-CCCCC3′), respectively. The minus indicates a control lacking enzyme. M specifies a size marker obtained by Orn-catalyzed reaction. Closed circle: 5-mers, open circle: 4-mers, closed triangle: 3-mers, open triangle: 2-mers, square: 1-mers.
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Figure 4: Comparison of YtqI and Orn-catalyzed degradation of nanoRNA 5-mers. Shown are the separation of reaction products on 22% PAA gels (upper panel) and the corresponding quantification (lower panel). Reactions contained 12 μg YtqI (A) or 0.14 μg Orn (B) and 1.5 μM or 2.7 μM RNA 5-mer (5′Cy5-CCCCC3′), respectively. The minus indicates a control lacking enzyme. M specifies a size marker obtained by Orn-catalyzed reaction. Closed circle: 5-mers, open circle: 4-mers, closed triangle: 3-mers, open triangle: 2-mers, square: 1-mers.

Mentions: Purified recombinant YtqI was tested for nanoRNase activity. In the presence of manganese, YtqI was able to degrade nanoRNA 5-mers (Figure 4). The activity in the presence of other ions tested (magnesium, zinc and calcium) was negligible (data not shown). Comparing YtqI- and Orn-catalyzed degradation of nanoRNA 5-mers, we noticed significant differences: The amount of YtqI required for appreciable activity was two orders of magnitude higher than that necessary for Orn-catalyzed activity. In addition, the pattern of degradation products as well as the kinetics of this reaction looked very different. Here, 3-mers were virtually missing and other intermediates (2-mers and 4-mers) accumulated less than in Orn-catalyzed hydrolysis (Figure 4). Therefore, we hypothesized that 3-mers might be a preferred substrate for YtqI and as such they might be hydrolyzed so fast that accumulation could not be observed. We tested this hypothesis by comparing degradation of 3-mers and 5-mers (Figures 4 and 5). We used three times more enzyme in the reaction with 5-mers as substrate in order to obtain appreciable conversion into monomers (Figure 4A) as compared to the reaction on 3-mers (Figure 5A). Turnover numbers for 3-mers were one order of magnitude higher than for 5-mers (1.5 versus 0.14 pmol/μg/min). In Figure 5B, we compare the kinetics of the disappearance of different substrates (3-mers or 5-mers) and the appearance of the final reaction product monomers in reactions with equal amounts of YtqI (1.5 μg). These results clearly document that 3-mers were a much better substrate for YtqI than 5-mers. Moreover, it seems that degradation of 3-mers to 2-mer was the fastest step in catalysis as the 2-mers formed here disappeared considerably slower.Figure 4.


YtqI from Bacillus subtilis has both oligoribonuclease and pAp-phosphatase activity.

Mechold U, Fang G, Ngo S, Ogryzko V, Danchin A - Nucleic Acids Res. (2007)

Comparison of YtqI and Orn-catalyzed degradation of nanoRNA 5-mers. Shown are the separation of reaction products on 22% PAA gels (upper panel) and the corresponding quantification (lower panel). Reactions contained 12 μg YtqI (A) or 0.14 μg Orn (B) and 1.5 μM or 2.7 μM RNA 5-mer (5′Cy5-CCCCC3′), respectively. The minus indicates a control lacking enzyme. M specifies a size marker obtained by Orn-catalyzed reaction. Closed circle: 5-mers, open circle: 4-mers, closed triangle: 3-mers, open triangle: 2-mers, square: 1-mers.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1935014&req=5

Figure 4: Comparison of YtqI and Orn-catalyzed degradation of nanoRNA 5-mers. Shown are the separation of reaction products on 22% PAA gels (upper panel) and the corresponding quantification (lower panel). Reactions contained 12 μg YtqI (A) or 0.14 μg Orn (B) and 1.5 μM or 2.7 μM RNA 5-mer (5′Cy5-CCCCC3′), respectively. The minus indicates a control lacking enzyme. M specifies a size marker obtained by Orn-catalyzed reaction. Closed circle: 5-mers, open circle: 4-mers, closed triangle: 3-mers, open triangle: 2-mers, square: 1-mers.
Mentions: Purified recombinant YtqI was tested for nanoRNase activity. In the presence of manganese, YtqI was able to degrade nanoRNA 5-mers (Figure 4). The activity in the presence of other ions tested (magnesium, zinc and calcium) was negligible (data not shown). Comparing YtqI- and Orn-catalyzed degradation of nanoRNA 5-mers, we noticed significant differences: The amount of YtqI required for appreciable activity was two orders of magnitude higher than that necessary for Orn-catalyzed activity. In addition, the pattern of degradation products as well as the kinetics of this reaction looked very different. Here, 3-mers were virtually missing and other intermediates (2-mers and 4-mers) accumulated less than in Orn-catalyzed hydrolysis (Figure 4). Therefore, we hypothesized that 3-mers might be a preferred substrate for YtqI and as such they might be hydrolyzed so fast that accumulation could not be observed. We tested this hypothesis by comparing degradation of 3-mers and 5-mers (Figures 4 and 5). We used three times more enzyme in the reaction with 5-mers as substrate in order to obtain appreciable conversion into monomers (Figure 4A) as compared to the reaction on 3-mers (Figure 5A). Turnover numbers for 3-mers were one order of magnitude higher than for 5-mers (1.5 versus 0.14 pmol/μg/min). In Figure 5B, we compare the kinetics of the disappearance of different substrates (3-mers or 5-mers) and the appearance of the final reaction product monomers in reactions with equal amounts of YtqI (1.5 μg). These results clearly document that 3-mers were a much better substrate for YtqI than 5-mers. Moreover, it seems that degradation of 3-mers to 2-mer was the fastest step in catalysis as the 2-mers formed here disappeared considerably slower.Figure 4.

Bottom Line: Firmicutes including Bacillus subtilis do not have an Oligoribonuclease (Orn) homologous protein and it is not yet understood which proteins accomplish the equivalent function in these organisms.An ytqI mutant in B. subtilis shows impairment of growth in the absence of cysteine, a phenotype resembling that of a cysQ mutant in E. coli.Phylogenetic distribution of YtqI, Orn and CysQ supports bifunctionality of YtqI.

View Article: PubMed Central - PubMed

Affiliation: Institut Pasteur, URA 2171, Unité de Génétique des Génomes Bactériens, 75724 Paris Cedex 15, France. umechold@pasteur.fr

ABSTRACT
Oligoribonuclease is the only RNase in Escherichia coli that is able to degrade RNA oligonucleotides five residues and shorter in length. Firmicutes including Bacillus subtilis do not have an Oligoribonuclease (Orn) homologous protein and it is not yet understood which proteins accomplish the equivalent function in these organisms. We had previously identified oligoribonucleases Orn from E. coli and its human homolog Sfn in a screen for proteins that are regulated by 3'-phosphoadenosine 5'-phosphate (pAp). Here, we identify YtqI as a potential functional analog of Orn through its interaction with pAp. YtqI degrades RNA oligonucleotides in vitro with preference for 3-mers. In addition, YtqI has pAp-phosphatase activity in vitro. In agreement with these data, YtqI is able to complement both orn and cysQ mutants in E. coli. An ytqI mutant in B. subtilis shows impairment of growth in the absence of cysteine, a phenotype resembling that of a cysQ mutant in E. coli. Phylogenetic distribution of YtqI, Orn and CysQ supports bifunctionality of YtqI.

Show MeSH
Related in: MedlinePlus