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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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Purification of a protein that inhibits DNA replication of an oriC-containing plasmid. (A) The indicated column fractions obtained by a Superdex 75 chromatography (GE Healthcare) were analyzed in a Coomassie blue-stained SDS–polyacrylamide gel (15% acrylamide). The elution volumes of monomeric DnaC (27.9 kDa) and other proteins were also determined for this column. (B) The amount of tL2 in (A) was quantified by densitometric analysis and expressed as net intensity, which is the background-subtracted pixel value for each band on the gel. (C) The extent of DNA synthesis was measured as described in ‘Materials and Methods’ section. DNA synthesis for an uninhibited reaction corresponds to about 320 pmol.
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Figure 1: Purification of a protein that inhibits DNA replication of an oriC-containing plasmid. (A) The indicated column fractions obtained by a Superdex 75 chromatography (GE Healthcare) were analyzed in a Coomassie blue-stained SDS–polyacrylamide gel (15% acrylamide). The elution volumes of monomeric DnaC (27.9 kDa) and other proteins were also determined for this column. (B) The amount of tL2 in (A) was quantified by densitometric analysis and expressed as net intensity, which is the background-subtracted pixel value for each band on the gel. (C) The extent of DNA synthesis was measured as described in ‘Materials and Methods’ section. DNA synthesis for an uninhibited reaction corresponds to about 320 pmol.

Mentions: Analysis of purified tL2 isolated in Figure 1 was performed with a Waters Q-TOF Ultima APT spectrometer coupled to a Waters Alliance 2795 HPLC system. Samples were loaded onto a 5 μ pore size 10 × 1 mm Beta Basic CN column, followed by gradient elution starting with 0.1% formic acid in water and ending with acetonitrile. Data were collected in the positive ion mode with a capillary voltage of 3 kV, with a source temperature of 90°C and a cone voltage of 35 eV.Figure 1.


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)

Purification of a protein that inhibits DNA replication of an oriC-containing plasmid. (A) The indicated column fractions obtained by a Superdex 75 chromatography (GE Healthcare) were analyzed in a Coomassie blue-stained SDS–polyacrylamide gel (15% acrylamide). The elution volumes of monomeric DnaC (27.9 kDa) and other proteins were also determined for this column. (B) The amount of tL2 in (A) was quantified by densitometric analysis and expressed as net intensity, which is the background-subtracted pixel value for each band on the gel. (C) The extent of DNA synthesis was measured as described in ‘Materials and Methods’ section. DNA synthesis for an uninhibited reaction corresponds to about 320 pmol.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 1: Purification of a protein that inhibits DNA replication of an oriC-containing plasmid. (A) The indicated column fractions obtained by a Superdex 75 chromatography (GE Healthcare) were analyzed in a Coomassie blue-stained SDS–polyacrylamide gel (15% acrylamide). The elution volumes of monomeric DnaC (27.9 kDa) and other proteins were also determined for this column. (B) The amount of tL2 in (A) was quantified by densitometric analysis and expressed as net intensity, which is the background-subtracted pixel value for each band on the gel. (C) The extent of DNA synthesis was measured as described in ‘Materials and Methods’ section. DNA synthesis for an uninhibited reaction corresponds to about 320 pmol.
Mentions: Analysis of purified tL2 isolated in Figure 1 was performed with a Waters Q-TOF Ultima APT spectrometer coupled to a Waters Alliance 2795 HPLC system. Samples were loaded onto a 5 μ pore size 10 × 1 mm Beta Basic CN column, followed by gradient elution starting with 0.1% formic acid in water and ending with acetonitrile. Data were collected in the positive ion mode with a capillary voltage of 3 kV, with a source temperature of 90°C and a cone voltage of 35 eV.Figure 1.

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