Requirements for and Regulation of Origin Opening of Plasmid P1

Park, Kyusung; Mukhopadhyay, Suman; Chattoraj, Dhruba K.

doi:10.1074/jbc.273.38.24906

Cited by 40 publications

(61 citation statements)

References 37 publications

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“…Because extra iterons reduce copy number efficiently, one of the mechanisms of copy number reduction thus could be by limiting new initiator synthesis. This view was supported by the finding that the copy number reduction by excess iterons could be overcome by increasing initiator concentration from constitutive sources (5,10,11). Thus, iteron-mediated copy number control in P1 plasmid may involve limiting RepA supply.…”

supporting

confidence: 64%

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Replication-induced transcription of an autorepressed gene: The replication initiator gene of plasmid P1

Mukhopadhyay¹,

Chattoraj²

2000

Proc. Natl. Acad. Sci. U.S.A.

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The replication origin of plasmid P1 contains an array of five repeats (iterons) that bind the plasmid-encoded initiator RepA. Within the array lies the repA promoter, which becomes largely repressed on RepA binding (autorepression). One might expect that extra iterons produced on plasmid replication would titrate RepA and release the repression. The promoter, however, is induced poorly by extra iterons. The P1 copy number is reduced by extra iterons in the presence of the autorepressed repA gene but not when additional RepA is provided from constitutive sources. It has been proposed that the iteron-bound RepA couples with the promoter-bound RepA and thereby maintains repression. Although not the product of replication, we find that the act of replication itself can renew RepA synthesis. Replication apparently cleans the promoter of bound RepA and provides a window of opportunity for repA transcription. We propose that replicationinduced transcription is required to ensure initiator availability in a system that is induced poorly when challenged with additional initiator binding sites.

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confidence: 64%

“…On several occasions, blockage of replication by extra iterons has been overcome by extra RepA supplied from foreign promoters (5,10,11). An intriguing feature of these experiments is that the extra RepA was required even though the autorepressed RepA source was present.…”

Section: Iterons Can Block Minip1mentioning

confidence: 99%

Replication-induced transcription of an autorepressed gene: The replication initiator gene of plasmid P1

Mukhopadhyay¹,

Chattoraj²

2000

Proc. Natl. Acad. Sci. U.S.A.

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“…It is possible that controlling RctB availability by rctA may become obligatory under some conditions. It is well known that a fewfold change in iteron and initiator concentrations can reverse their activities from stimulatory to inhibitory in iteron-carrying plasmids (35).…”

Section: Discussionmentioning

confidence: 99%

Transcriptional inactivation of a regulatory site for replication of Vibrio cholerae chromosome II

Venkova-Canova¹,

Srivastava²,

Chattoraj³

2006

Proc. Natl. Acad. Sci. U.S.A.

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The oriCII region that includes the initiator gene, rctB, can function as a plasmid in E. coli. Here we show that RctB suffices for the oriCII-based plasmid replication, and rctA in cis or trans reduces the plasmid copy number, thereby serving as a negative regulator. The inhibitory activity could be overcome by increasing the concentration of RctB, suggesting that rctA titrates the initiator. Purified RctB bound to a DNA fragment carrying rctA, confirming that the two can interact. Although rctA apparently works as a titrating site, it is nonetheless transcribed. We find that the transcription attenuates the inhibitory activity of the gene, presumably by interfering with RctB binding. RctB, in turn, repressed the rctA promoter and, thereby, could control its own titration by modulating the transcription of rctA. This control circuit appears to be a putative novel mechanism for homeostasis of initiator availability.O ur knowledge of DNA replication control in bacteria comes largely from studying the chromosome and plasmids of Escherichia coli (1, 2). In general, controlling proteins that initiate replication, initiator proteins, seems to be the central mechanism to regulate replication. A variety of mechanisms have been found to control the initiator.Transcriptional autoregulation of the initiator gene is common in plasmids with iterated initiator-binding sites (iterons), where the initiator serves as the repressor of its own synthesis (3). The E. coli initiator, DnaA, also is autoregulated (4). In addition, the dnaA promoter is inactivated by sequestration to the cell membrane for approximately one-third of the cell generation (5). More traditional modes of transcriptional control using repressors and activators also have been found in phage and plasmid RK2 (6, 7).Initiator translation is regulated by antisense RNAs in a variety of plasmids, the classic examples being plasmids R1 and pT181 (8, 9). The antisense RNA either blocks the ribosomebinding site of the initiator gene directly or of a regulatory peptide for the initiator gene (10).Most other mechanisms operate posttranslationally either by titrating or inactivating the initiator protein. In E. coli, there are Ϸ300 DnaA-binding sites scattered all over the chromosome (11,12). Titration of the initiator to these sites is believed to reduce availability of the protein after replication initiation, because new sites are created during replication elongation.Initiator inactivation is the most widely used mechanism to prevent premature replication reinitiation. The pT181 initiator is inactivated by covalent modification at the end of one round of replication (13). E. coli DnaA is converted from an active ATP bound form to an inactive ADP form after replication (14-16). A similar mechanism also operates in Bacillus subtilis (17). The initiators of iteron-carrying plasmids are inactivated by dimerization, the active form being monomers (18). Dimerization not only reduces monomer concentration, the dimers also participate in inactivating origins (19)(20)(21). Euk...

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“…Thus, although Rep proteins can promote some structural transitions in DNA, most plasmids require DnaA to aid in origin melting (17,(25)(26)(27)(28)(29)(30) and helicase loading (28,29). However, they still experience conformational activation (reviewed in Ref.…”

mentioning

confidence: 99%

Structural Changes in RepA, a Plasmid Replication Initiator, upon Binding to Origin DNA

Díaz-López

Lages-Gonzalo

Serrano-López

et al. 2003

Journal of Biological Chemistry

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RepA protein is the DNA replication initiator of the Pseudomonas plasmid pPS10. RepA dimers bind to an inversely repeated operator sequence in repA promoter, thus repressing its own synthesis, whereas monomers bind to four directly repeated sequences (iterons) to initiate DNA replication. We had proposed previously that RepA is composed of two winged-helix (WH) domains, a structural unit also present in eukaryotic and archaeal initiators. To bind to the whole iteron sequence through both domains, RepA should couple monomerization to a conformational change in the N-terminal WH, which includes a leucine zipper-like sequence motif. We show for the first time that, by itself, binding to iteron DNA in vitro dissociates RepA dimers into monomers and alters RepA conformation, suggesting an allosteric effect. Furthermore, we also show that similar changes in RepA are promoted by mutations that substitute two Leu residues of the putative leucine zipper by Ala, destabilizing the hydrophobic core of the first WH. We propose that this mutant (RepA-2L2A) resembles a transient folding intermediate in the pathway leading to active monomers. These findings, together with the known activation of other Rep-type proteins by chaperones, are relevant to understand the molecular basis of plasmid DNA replication initiation.

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Requirements for and Regulation of Origin Opening of Plasmid P1

Cited by 40 publications

References 37 publications

Replication-induced transcription of an autorepressed gene: The replication initiator gene of plasmid P1

Replication-induced transcription of an autorepressed gene: The replication initiator gene of plasmid P1

Transcriptional inactivation of a regulatory site for replication of Vibrio cholerae chromosome II

Structural Changes in RepA, a Plasmid Replication Initiator, upon Binding to Origin DNA

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