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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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Both forms of L2 require the N-terminal region of DnaA to interact. In (A), the indicated amounts of DnaA or galectin 3 were added in triplicate to the wells containing immobilized tL2 (100 ng), L2 (100 ng) or HU (590 ng; α dimer). The vertical lines indicate the standard deviation. For comparison with the plot of DnaA retained by immobilized HU or tL2 or L2, the plot labeled ‘DnaA only’ represents the amount of DnaA (6, 12, 25, 50 and 100 ng) that was immobilized directly to the microtiter plate (see ‘Materials and Methods’ section). In (B), the column labeled ‘Retention Alone’ reflects the amount of DnaA or the respective mutant DnaA (each at 50 ng) that was immobilized when added directly to the microtiter plate. The columns labeled ‘Retention by HU (α2)’, ‘Retention by L2’ and ‘Retention by tL2’ compare the retention of DnaA or the different mutant DnaAs (each at 50 ng) by immobilized HU (α dimer), L2 or tL2, respectively. The columns labeled ‘Ratio’ represent the normalized absorbance relative to wild-type DnaA, which was set at 1.
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Figure 7: Both forms of L2 require the N-terminal region of DnaA to interact. In (A), the indicated amounts of DnaA or galectin 3 were added in triplicate to the wells containing immobilized tL2 (100 ng), L2 (100 ng) or HU (590 ng; α dimer). The vertical lines indicate the standard deviation. For comparison with the plot of DnaA retained by immobilized HU or tL2 or L2, the plot labeled ‘DnaA only’ represents the amount of DnaA (6, 12, 25, 50 and 100 ng) that was immobilized directly to the microtiter plate (see ‘Materials and Methods’ section). In (B), the column labeled ‘Retention Alone’ reflects the amount of DnaA or the respective mutant DnaA (each at 50 ng) that was immobilized when added directly to the microtiter plate. The columns labeled ‘Retention by HU (α2)’, ‘Retention by L2’ and ‘Retention by tL2’ compare the retention of DnaA or the different mutant DnaAs (each at 50 ng) by immobilized HU (α dimer), L2 or tL2, respectively. The columns labeled ‘Ratio’ represent the normalized absorbance relative to wild-type DnaA, which was set at 1.

Mentions: In other work, we discovered that a DnaA affinity column and not a BSA column specifically retained L2 from a soluble lysate (Vicente and Kaguni, data not shown). DnaA was also found to interact with L2 via a proteomics approach (47). To extend these observations, we showed in a solid phase binding assay that tL2 and L2 interacts with wild-type DnaA, or a mutant DnaA (DnaAΔ220-294) lacking residues 220–294, but that this interaction was reduced by about 4-fold with DnaAΔ129 lacking the N-terminal 129 amino acids (Figure 7). DnaAΔ129 was also defective in interaction with HU, confirming previous results (9). We know that the preparation of DnaAΔ129 is as active as DnaA+ in oriC unwinding, and in binding to ATP or to an oriC-containing DNA fragment (20,35), so the mutant DnaA is not grossly misfolded. As negative controls, we showed that tL2 or L2 does not bind to DnaB (data not shown) or murine galectin-3 (Figure 7A), which is involved in pre-mRNA splicing and is not expected to interact (48). These results suggest that both forms of L2 inhibit DNA replication of the oriC-containing plasmid by interacting with the N-terminal region of DnaA. As described above, it is possible that their non-specific DNA binding activity may also contribute to the inhibition.Figure 7.


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)

Both forms of L2 require the N-terminal region of DnaA to interact. In (A), the indicated amounts of DnaA or galectin 3 were added in triplicate to the wells containing immobilized tL2 (100 ng), L2 (100 ng) or HU (590 ng; α dimer). The vertical lines indicate the standard deviation. For comparison with the plot of DnaA retained by immobilized HU or tL2 or L2, the plot labeled ‘DnaA only’ represents the amount of DnaA (6, 12, 25, 50 and 100 ng) that was immobilized directly to the microtiter plate (see ‘Materials and Methods’ section). In (B), the column labeled ‘Retention Alone’ reflects the amount of DnaA or the respective mutant DnaA (each at 50 ng) that was immobilized when added directly to the microtiter plate. The columns labeled ‘Retention by HU (α2)’, ‘Retention by L2’ and ‘Retention by tL2’ compare the retention of DnaA or the different mutant DnaAs (each at 50 ng) by immobilized HU (α dimer), L2 or tL2, respectively. The columns labeled ‘Ratio’ represent the normalized absorbance relative to wild-type DnaA, which was set at 1.
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Figure 7: Both forms of L2 require the N-terminal region of DnaA to interact. In (A), the indicated amounts of DnaA or galectin 3 were added in triplicate to the wells containing immobilized tL2 (100 ng), L2 (100 ng) or HU (590 ng; α dimer). The vertical lines indicate the standard deviation. For comparison with the plot of DnaA retained by immobilized HU or tL2 or L2, the plot labeled ‘DnaA only’ represents the amount of DnaA (6, 12, 25, 50 and 100 ng) that was immobilized directly to the microtiter plate (see ‘Materials and Methods’ section). In (B), the column labeled ‘Retention Alone’ reflects the amount of DnaA or the respective mutant DnaA (each at 50 ng) that was immobilized when added directly to the microtiter plate. The columns labeled ‘Retention by HU (α2)’, ‘Retention by L2’ and ‘Retention by tL2’ compare the retention of DnaA or the different mutant DnaAs (each at 50 ng) by immobilized HU (α dimer), L2 or tL2, respectively. The columns labeled ‘Ratio’ represent the normalized absorbance relative to wild-type DnaA, which was set at 1.
Mentions: In other work, we discovered that a DnaA affinity column and not a BSA column specifically retained L2 from a soluble lysate (Vicente and Kaguni, data not shown). DnaA was also found to interact with L2 via a proteomics approach (47). To extend these observations, we showed in a solid phase binding assay that tL2 and L2 interacts with wild-type DnaA, or a mutant DnaA (DnaAΔ220-294) lacking residues 220–294, but that this interaction was reduced by about 4-fold with DnaAΔ129 lacking the N-terminal 129 amino acids (Figure 7). DnaAΔ129 was also defective in interaction with HU, confirming previous results (9). We know that the preparation of DnaAΔ129 is as active as DnaA+ in oriC unwinding, and in binding to ATP or to an oriC-containing DNA fragment (20,35), so the mutant DnaA is not grossly misfolded. As negative controls, we showed that tL2 or L2 does not bind to DnaB (data not shown) or murine galectin-3 (Figure 7A), which is involved in pre-mRNA splicing and is not expected to interact (48). These results suggest that both forms of L2 inhibit DNA replication of the oriC-containing plasmid by interacting with the N-terminal region of DnaA. As described above, it is possible that their non-specific DNA binding activity may also contribute to the inhibition.Figure 7.

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