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Two forms of ribosomal protein L2 of Escherichia coli that inhibit DnaA in DNA replication.

Chodavarapu S, Felczak MM, Kaguni JM - Nucleic Acids Res. (2011)

Bottom Line: We purified an inhibitor of oriC plasmid replication and determined that it is a truncated form of ribosomal protein L2 evidently lacking 59 amino acid residues from the C-terminal region encoded by rplB.We show that this truncated form of L2 or mature L2 physically interacts with the N-terminal region of DnaA to inhibit initiation from oriC by apparently interfering with DnaA oligomer formation, and the subsequent assembly of the prepriming complex on an oriC plasmid.Both forms of L2 also inhibit the unwinding of oriC by DnaA.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824-1319, USA.

ABSTRACT
We purified an inhibitor of oriC plasmid replication and determined that it is a truncated form of ribosomal protein L2 evidently lacking 59 amino acid residues from the C-terminal region encoded by rplB. We show that this truncated form of L2 or mature L2 physically interacts with the N-terminal region of DnaA to inhibit initiation from oriC by apparently interfering with DnaA oligomer formation, and the subsequent assembly of the prepriming complex on an oriC plasmid. Both forms of L2 also inhibit the unwinding of oriC by DnaA. These in vitro results raise the possibility that one or both forms of L2 modulate DnaA function in vivo to regulate the frequency of initiation.

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tL2 and L2 destabilize DnaA bound to oriC. The prepriming complex was assembled as described in ‘Materials and Methods’ section. After isolation of the complex, the amounts of DnaA and DnaB in the complex assembled in the presence or absence of tL2 (A) or L2 (C) were measured by immunoblot analysis. In (B) and (D), the activity of the isolated prepriming complex was measured by the addition of required reagents and proteins needed for DNA synthesis to 20 µl of the indicated column fractions. After adjusting the concentration of magnesium acetate to 10 mM in a reaction volume of 25 µl, DNA synthesis was measured. Where indicated, tL2 (190 ng) or L2 (180 ng) was added to determine its effect on the isolated prepriming complex that was assembled in its absence.
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Figure 8: tL2 and L2 destabilize DnaA bound to oriC. The prepriming complex was assembled as described in ‘Materials and Methods’ section. After isolation of the complex, the amounts of DnaA and DnaB in the complex assembled in the presence or absence of tL2 (A) or L2 (C) were measured by immunoblot analysis. In (B) and (D), the activity of the isolated prepriming complex was measured by the addition of required reagents and proteins needed for DNA synthesis to 20 µl of the indicated column fractions. After adjusting the concentration of magnesium acetate to 10 mM in a reaction volume of 25 µl, DNA synthesis was measured. Where indicated, tL2 (190 ng) or L2 (180 ng) was added to determine its effect on the isolated prepriming complex that was assembled in its absence.

Mentions: Recent work indicates that HU or DiaA stabilizes the DnaA oligomer by interacting with DnaA’s N-terminal region (9,10), which acts in self-oligomerization (4,26). We considered the converse possibility that both forms of L2 inhibit initiation by destabilizing DnaA oligomerized at oriC. To test this idea, we assembled the prepriming complex in the presence and absence of tL2 or L2 as described (6,26). After separating the complex from unbound proteins by gel filtration chromatography, we quantified the amounts of DnaA and DnaB in the complex by immunoblotting relative to a standard curve prepared with known amounts of each protein (Figure 8). We quantified DnaC in a separate study (7). Relative to the amount of supercoiled oriC plasmid in the complex (data not shown), both ribosomal proteins led to reduced amounts of DnaA and DnaB in the isolated complex. Hence, the interaction of tL2 or L2 with DnaA’s N-terminal region apparently destabilizes the DnaA oligomer to inhibit the loading of DnaB at oriC.Figure 8.


Two forms of ribosomal protein L2 of Escherichia coli that inhibit DnaA in DNA replication.

Chodavarapu S, Felczak MM, Kaguni JM - Nucleic Acids Res. (2011)

tL2 and L2 destabilize DnaA bound to oriC. The prepriming complex was assembled as described in ‘Materials and Methods’ section. After isolation of the complex, the amounts of DnaA and DnaB in the complex assembled in the presence or absence of tL2 (A) or L2 (C) were measured by immunoblot analysis. In (B) and (D), the activity of the isolated prepriming complex was measured by the addition of required reagents and proteins needed for DNA synthesis to 20 µl of the indicated column fractions. After adjusting the concentration of magnesium acetate to 10 mM in a reaction volume of 25 µl, DNA synthesis was measured. Where indicated, tL2 (190 ng) or L2 (180 ng) was added to determine its effect on the isolated prepriming complex that was assembled in its absence.
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Related In: Results  -  Collection

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Figure 8: tL2 and L2 destabilize DnaA bound to oriC. The prepriming complex was assembled as described in ‘Materials and Methods’ section. After isolation of the complex, the amounts of DnaA and DnaB in the complex assembled in the presence or absence of tL2 (A) or L2 (C) were measured by immunoblot analysis. In (B) and (D), the activity of the isolated prepriming complex was measured by the addition of required reagents and proteins needed for DNA synthesis to 20 µl of the indicated column fractions. After adjusting the concentration of magnesium acetate to 10 mM in a reaction volume of 25 µl, DNA synthesis was measured. Where indicated, tL2 (190 ng) or L2 (180 ng) was added to determine its effect on the isolated prepriming complex that was assembled in its absence.
Mentions: Recent work indicates that HU or DiaA stabilizes the DnaA oligomer by interacting with DnaA’s N-terminal region (9,10), which acts in self-oligomerization (4,26). We considered the converse possibility that both forms of L2 inhibit initiation by destabilizing DnaA oligomerized at oriC. To test this idea, we assembled the prepriming complex in the presence and absence of tL2 or L2 as described (6,26). After separating the complex from unbound proteins by gel filtration chromatography, we quantified the amounts of DnaA and DnaB in the complex by immunoblotting relative to a standard curve prepared with known amounts of each protein (Figure 8). We quantified DnaC in a separate study (7). Relative to the amount of supercoiled oriC plasmid in the complex (data not shown), both ribosomal proteins led to reduced amounts of DnaA and DnaB in the isolated complex. Hence, the interaction of tL2 or L2 with DnaA’s N-terminal region apparently destabilizes the DnaA oligomer to inhibit the loading of DnaB at oriC.Figure 8.

Bottom Line: We purified an inhibitor of oriC plasmid replication and determined that it is a truncated form of ribosomal protein L2 evidently lacking 59 amino acid residues from the C-terminal region encoded by rplB.We show that this truncated form of L2 or mature L2 physically interacts with the N-terminal region of DnaA to inhibit initiation from oriC by apparently interfering with DnaA oligomer formation, and the subsequent assembly of the prepriming complex on an oriC plasmid.Both forms of L2 also inhibit the unwinding of oriC by DnaA.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824-1319, USA.

ABSTRACT
We purified an inhibitor of oriC plasmid replication and determined that it is a truncated form of ribosomal protein L2 evidently lacking 59 amino acid residues from the C-terminal region encoded by rplB. We show that this truncated form of L2 or mature L2 physically interacts with the N-terminal region of DnaA to inhibit initiation from oriC by apparently interfering with DnaA oligomer formation, and the subsequent assembly of the prepriming complex on an oriC plasmid. Both forms of L2 also inhibit the unwinding of oriC by DnaA. These in vitro results raise the possibility that one or both forms of L2 modulate DnaA function in vivo to regulate the frequency of initiation.

Show MeSH
Related in: MedlinePlus