Limits...
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.

Show MeSH

Related in: MedlinePlus

Compared with L2, tL2 does not sequester the supercoils of a plasmid DNA. The change in DNA topology of a supercoiled plasmid was measured as described in ‘Materials and Methods’ section. The figure shows the reverse contrast image of DNAs with different superhelix densities that were visualized by ethidium bromide fluorescence.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3105425&req=5

Figure 4: Compared with L2, tL2 does not sequester the supercoils of a plasmid DNA. The change in DNA topology of a supercoiled plasmid was measured as described in ‘Materials and Methods’ section. The figure shows the reverse contrast image of DNAs with different superhelix densities that were visualized by ethidium bromide fluorescence.

Mentions: Because DNA binding proteins such as HU or FIS at an equal weight ratio to DNA inhibit oriC plasmid replication by sequestering its negative superhelicity (39–41), we considered whether this mechanism could explain the inhibitory effect of the ribosomal proteins. To test this possibility, we incubated the oriC plasmid with increasing amounts of either form of L2, and included a topoisomerase to stabilize any topological alteration followed by electrophoresis in an agarose gel containing chloroquine. Like HU, we found that L2 but not tL2 sequestered the negative supercoils of the oriC plasmid in proportion with the amount added (Figure 4), which could explain L2′s ability to inhibit DNA replication of the oriC plasmid. However, the results described below suggest that both forms of L2 interfere with initiation in vitro by an independent mechanism.Figure 4.


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)

Compared with L2, tL2 does not sequester the supercoils of a plasmid DNA. The change in DNA topology of a supercoiled plasmid was measured as described in ‘Materials and Methods’ section. The figure shows the reverse contrast image of DNAs with different superhelix densities that were visualized by ethidium bromide fluorescence.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3105425&req=5

Figure 4: Compared with L2, tL2 does not sequester the supercoils of a plasmid DNA. The change in DNA topology of a supercoiled plasmid was measured as described in ‘Materials and Methods’ section. The figure shows the reverse contrast image of DNAs with different superhelix densities that were visualized by ethidium bromide fluorescence.
Mentions: Because DNA binding proteins such as HU or FIS at an equal weight ratio to DNA inhibit oriC plasmid replication by sequestering its negative superhelicity (39–41), we considered whether this mechanism could explain the inhibitory effect of the ribosomal proteins. To test this possibility, we incubated the oriC plasmid with increasing amounts of either form of L2, and included a topoisomerase to stabilize any topological alteration followed by electrophoresis in an agarose gel containing chloroquine. Like HU, we found that L2 but not tL2 sequestered the negative supercoils of the oriC plasmid in proportion with the amount added (Figure 4), which could explain L2′s ability to inhibit DNA replication of the oriC plasmid. However, the results described below suggest that both forms of L2 interfere with initiation in vitro by an independent mechanism.Figure 4.

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