Biomedical subjects

Katarzyna Potrykus

Publications and source records attributed to Katarzyna Potrykus.

4 recordsLinked to original sources

PubMed2002

Multiple mechanisms of transcription inhibition by ppGpp at the lambdap(R) promoter.

General stress conditions in bacterial cells cause a global cellular response called the stringent response. The first event in this control is production of large amounts of a regulatory nucleotide, guanosine-3',5'-(bis)pyrophospahte (ppGpp). It was proposed recently that ppGpp acts by decreasing stability of open complexes at promoters that make short-lived open complexes, e.g. the rRNA promoters. However, here we report that the bacteriophage lambdap(R) promoter, which forms long-lived open complexes, is inhibited by ppGpp in vitro as observed in vivo. We performed a systematic investigation of the ppGpp-specific inhibition of transcription initiation at lambdap(R) and found that ppGpp does decrease stability of open complexes at lambdap(R), but only slightly. Likewise the equilbrium binding constant and rate of open complex formation by RNA polymerase at lambdap(R) are only slightly affected by ppGpp. The major effect of ppGpp-mediated inhibition is to decrease the rate of promoter escape. We conclude that ppGpp-mediated inhibition of transcription initiation is not restricted to promoters that make short-lived open complexes. Rather we conclude that the initial catalytic step of transcript formation is affected by ppGpp, specifically formation of the first phosphodiester bond is inhibited by ppGpp at lambdap(R).

Blotting, Northern↗

PubMed2002

Composition of the lambda plasmid heritable replication complex.

Previous studies indicated during replication of plasmids derived from bacteriophage lambda (the so-called lambda plasmids), that, once assembled, replication complex can be inherited by one of the two daughter plasmid copies after each replication round, and may function in subsequent replication rounds. It seems that similar processes occur during replication of other DNA molecules, including chromosomes of the yeast Saccharomyces cerevisiae. However, apart from some suggestions based on genetic experiments, composition of the lambda heritable replication complex remains unknown. In amino acid-starved Escherichia coli relA mutants, replication of lambda plasmid DNA is carried out exclusively by the heritable replication complex as assembly of new complexes is impaired due to inhibition of protein synthesis. Here, using a procedure based on in vivo cross-linking, cell lysis, immunoprecipitation with specific sera, de-cross-linking and PCR analysis, we demonstrate that the lambda heritable replication complex consists of O, P, DnaB and, perhaps surprisingly, DnaK proteins.

Base Sequence↗

PubMed2002

Lambdap(o), a promoter for oop RNA synthesis, has a role in replication of plasmids derived from bacteriophage lambda.

Transcription initiated at the bacteriophage lambdap(o) promoter gives a short RNA, called oop RNA. Early studies led to a proposal that this transcript plays a role in the initiation of lambda DNA replication. In fact, the p(o) promoter is located in the lambda replication region and it was suggested that oop RNA may be a primer for replication proceeding leftward from orilambda. However, since in vitro experiments demonstrated that primers for lambda DNA replication are produced by the dnaG gene product (DnaG primase) and subsequent in vivo studies indicated that oop RNA is an antisense RNA for the lambda cII gene expression, the above-mentioned hypothesis has fallen into oblivion. Nevertheless, here we demonstrate that the p(o) promoter plays a role in lambda DNA replication, indeed. We found that lambda plasmids bearing a mutation that inactivates p(o) occur in Escherichia coli cells in a copy number significantly lower than wild-type lambda plasmids. Amplification of lambdap(o)(-) plasmids during the relaxed response was less efficient relative to lambdap(o)(+) plasmids suggesting less frequent initiation of replication from orilambda in the absence of transcription from p(o). This suggestion was confirmed by measurement of incorporation of [(3)H]thymidine into lambda plasmid DNA during pulse-labeling experiments. Therefore, we propose that transcription from the p(o) promoter stimulates replication initiation at orilambda as suggested a long time ago, however, contrary to that suggestion, we assume that the process of p(o)-initiated transcription per se but not the transcription product (oop RNA) might play a role at early steps of lambda DNA replication.

Bacteriophage lambda↗

PubMed2002

Differential effects of Kid toxin on two modes of replication of lambdoid plasmids suggest that this toxin acts before, but not after, the assembly of the replication complex.

Kid is a small protein that is encoded by plasmid R1. It is a toxin that belongs to a killer system that ensures the stability of the plasmid in host cells. The results of previous studies have suggested that Kid is an inhibitor of DNA replication, possibly acting at the onset of initiation. Here, the authors tested the effects of Kid on orilambda-intitiated and oriJ-initiated replication, which may be driven by both the newly assembled replication complex and the heritable complex. It was found that Kid inhibits only replication that is driven by the newly assembled replication complex. The authors also report that Kid inhibits ColE1-like plasmid replication in vivo, in agreement with the previously reported inhibition of ColE1 during in vitro replication. It is proposed that the Kid toxin acts at the level of replication either by preventing de novo assembly of the replication complex or by impairing the functional interactions of the replication complex at the initiation stage.

Bacterial Proteins↗