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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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Immunoblot analysis reveals that the purified protein is a truncated form of L2, which exists in log phase cells. In (A), L2 (25 ng) from the Nierhaus laboratory, purified tL2 (15 ng) and whole-cell lysates from stationary (0.4 OD595nm) and log phase cells (0.3 OD595nm) were analyzed by immunoblotting with affinity-purified polyclonal antibody that specifically recognizes L2. Electrophoretic separation was in a 15% SDS–polyacrylamide gel. In (B), electrospray ionization mass spectrometry of purified tL2 isolated in Figure 1 was performed as described in ‘Materials and Methods’ section.
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Figure 2: Immunoblot analysis reveals that the purified protein is a truncated form of L2, which exists in log phase cells. In (A), L2 (25 ng) from the Nierhaus laboratory, purified tL2 (15 ng) and whole-cell lysates from stationary (0.4 OD595nm) and log phase cells (0.3 OD595nm) were analyzed by immunoblotting with affinity-purified polyclonal antibody that specifically recognizes L2. Electrophoretic separation was in a 15% SDS–polyacrylamide gel. In (B), electrospray ionization mass spectrometry of purified tL2 isolated in Figure 1 was performed as described in ‘Materials and Methods’ section.

Mentions: We confirmed the identity of the polypeptide by immunoblot analysis with an affinity-purified antibody that reacts specifically with L2 (Figure 2A). However, the polypeptide isolated has an electrophoretic mobility that is greater than L2 obtained as a gift from the Nierhaus laboratory. Its position is very similar to one of two polypeptides in a whole-cell lysate of a log phase culture of E. coli DM700 from which we purified this form of L2, but only the larger L2 was detected in a stationary phase culture. We also detected this truncated form of L2 (tL2) in log phase cultures of E. coli MG1655 and W3110 (data not shown), suggesting that tL2 is present in many other E. coli strains. Hence, tL2 does not apparently arise by proteolysis during purification. These findings raise the possibility that L2, like other ribosomal proteins summarized in the ‘Discussion’ section, has dual roles. We do not know if all log phase cells contain a basal level or if tL2 only appears in a subpopulation of cells in response to a signal (see ‘Discussion’ section).Figure 2.


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)

Immunoblot analysis reveals that the purified protein is a truncated form of L2, which exists in log phase cells. In (A), L2 (25 ng) from the Nierhaus laboratory, purified tL2 (15 ng) and whole-cell lysates from stationary (0.4 OD595nm) and log phase cells (0.3 OD595nm) were analyzed by immunoblotting with affinity-purified polyclonal antibody that specifically recognizes L2. Electrophoretic separation was in a 15% SDS–polyacrylamide gel. In (B), electrospray ionization mass spectrometry of purified tL2 isolated in Figure 1 was performed as described in ‘Materials and Methods’ section.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 2: Immunoblot analysis reveals that the purified protein is a truncated form of L2, which exists in log phase cells. In (A), L2 (25 ng) from the Nierhaus laboratory, purified tL2 (15 ng) and whole-cell lysates from stationary (0.4 OD595nm) and log phase cells (0.3 OD595nm) were analyzed by immunoblotting with affinity-purified polyclonal antibody that specifically recognizes L2. Electrophoretic separation was in a 15% SDS–polyacrylamide gel. In (B), electrospray ionization mass spectrometry of purified tL2 isolated in Figure 1 was performed as described in ‘Materials and Methods’ section.
Mentions: We confirmed the identity of the polypeptide by immunoblot analysis with an affinity-purified antibody that reacts specifically with L2 (Figure 2A). However, the polypeptide isolated has an electrophoretic mobility that is greater than L2 obtained as a gift from the Nierhaus laboratory. Its position is very similar to one of two polypeptides in a whole-cell lysate of a log phase culture of E. coli DM700 from which we purified this form of L2, but only the larger L2 was detected in a stationary phase culture. We also detected this truncated form of L2 (tL2) in log phase cultures of E. coli MG1655 and W3110 (data not shown), suggesting that tL2 is present in many other E. coli strains. Hence, tL2 does not apparently arise by proteolysis during purification. These findings raise the possibility that L2, like other ribosomal proteins summarized in the ‘Discussion’ section, has dual roles. We do not know if all log phase cells contain a basal level or if tL2 only appears in a subpopulation of cells in response to a signal (see ‘Discussion’ section).Figure 2.

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