Coupling of transcription and replication machineries in the λ DNA replication initiation: evidence for direct interaction of Escherichia coli RNA polymerase and the lambda O protein

Transcription proceeding downstream of the lambda phage replication origin was previously shown to support initial steps of the lambda primosome assembly in vitro and to regulate frequency and directionality of lambda DNA replication in vivo. In this report, the data are presented indicating that the RNA polymerase beta subunit makes a direct contact with the lambdaO protein, a replication initiator of lambda phage. These results suggest that the role of RNA polymerase during the initiation of lambda phage DNA replication may be more complex than solely influencing DNA topology. Results demonstrated in this study also show that gyrase supercoiling activity stimulates the formation of a complex between lambdaO and RNA polymerase, suggesting that the introduction of negative supercoils by DNA gyrase, besides lowering the energy required for DNA strand separation, may play an additional role in modeling protein–protein interactions at early steps of DNA replication initiation

Wprowadzenie

Wprowadzenie do nr 270 Acta Universitatis Lodziensis, Folia Oeconomic

Toxicity of the bacteriophage λ cII gene product to Escherichia coli arises from inhibition of host cell DNA replication

AbstractThe bacteriophage λ cII gene codes for a transcriptional activator protein which is a crucial regulator at the stage of the “lysis-versus-lysogeny” decision during phage development. The CII protein is highly toxic to the host, Escherichia coli, when overproduced. However, the molecular mechanism of this toxicity is not known. Here we demonstrate that DNA synthesis, but not total RNA synthesis, is strongly inhibited in cII-overexpressing E. coli cells. The toxicity was also observed when the transcriptional stimulator activity of CII was abolished either by a point mutation in the cII gene or by a point mutation, rpoA341, in the gene coding for the RNA polymerase α subunit. Moreover, inhibition of cell growth, caused by both wild-type and mutant CII proteins in either rpoA+ or rpoA341 hosts, could be relieved by overexpression of the E. coli dnaB and dnaC genes. In vitro replication of an oriC-based plasmid DNA was somewhat impaired by the presence of the CII, and several CII-resistant E. coli strains contain mutations near dnaC. We conclude that the DNA replication machinery may be a target for the toxic activity of CII

Zarządzanie przedsięwzięciami budowlanymi w gospodarce polskiej: podstawy, procedury, przykłady

Zdjęcia w tekście: Jarosław Hałuszczak, Inwestycja II linii Metra w Warszawie, Inwestycja Złote Tarasy, Inwestycja Gemini Park, Inwestycja Tryton Park, Inwestycja GDDKiA, Inwestycja Autostrada Wielkopolska II – GDDKiA, Marcin Jurek, Inwestycje PGNiG SA, Elżbieta StrzeleckaProjekt okładki: Studio GKDS, www.gkds.pl; w tym zdjęcia: flickr.com, Wojciech Klemm – Inwestycja UNIBUD Sp. z o.o. w Łodzi, Photogenica.pl, Elżbieta Strzelecka – Politechnika Łódzka w ŁodziCelem publikacji jest przedstawienie wybranych zagadnień dotyczących zarządzania przedsięwzięciami budowlanymi na krajowym rynku budowlanym z uwzględnieniem aspektów technicznych, technologicznych, organizacyjnych, ekonomicznych, społecznych i środowiskowych. Podręcznik ten adresowany jest do studentów, menedżerów, praktyków związanych z budowlanymi projektami inwestycyjnymi. Zagadnienia poruszane w nim mogą stanowić ułatwienie w studiowaniu problematyki kierowania procesem inwestycyjnym, organizacji procesu inwestycyjnego, planowania i finansowania procesów inwestycyjnych, zarządzania ryzykiem, zarządzania jakością w procesach budowlanych, prawnych uwarunkowań procesów inwestycyjnych i ich wpływu na planowanie przestrzenne, zrównoważonego rozwoju w budownictwie

Coupling of transcription and replication machineries in λ DNA replication initiation: evidence for direct interaction of Escherichia coli RNA polymerase and the λO protein

Transcription proceeding downstream of the λ phage replication origin was previously shown to support initial steps of the λ primosome assembly in vitro and to regulate frequency and directionality of λ DNA replication in vivo. In this report, the data are presented indicating that the RNA polymerase β subunit makes a direct contact with the λO protein, a replication initiator of λ phage. These results suggest that the role of RNA polymerase during the initiation of λ phage DNA replication may be more complex than solely influencing DNA topology. Results demonstrated in this study also show that gyrase supercoiling activity stimulates the formation of a complex between λO and RNA polymerase, suggesting that the introduction of negative supercoils by DNA gyrase, besides lowering the energy required for DNA strand separation, may play an additional role in modeling protein–protein interactions at early steps of DNA replication initiation

Influence of the Escherichia coli oxyR gene function on λ prophage maintenance

In Escherichia coli hosts, hydrogen peroxide is one of the factors that may cause induction of λ prophage. Here, we demonstrate that H2O2-mediated λ prophage induction is significantly enhanced in the oxyR mutant host. The mRNA levels for cI gene expression were increased in a λ lysogen in the presence of H2O2. On the other hand, stimulation of the pM promoter by cI857 overproduced from a multicopy plasmid was decreased in the ΔoxyR mutant in the presence of H2O2 but not under normal growth conditions. The purified OxyR protein did bind specifically to the pM promoter region. This binding impaired efficiency of interaction of the cI protein with the OR3 site, while stimulating such a binding to OR2 and OR1 sites, in the regulatory region of the pM promoter. We propose that changes in cI gene expression, perhaps in combination with moderately induced SOS response, may be responsible for enhanced λ prophage induction by hydrogen peroxide in the oxyR mutant. Therefore, OxyR seems to be a factor stimulating λ prophage maintenance under conditions of oxidative stress. This proposal is discussed in the light of efficiency of induction of lambdoid prophages bearing genes coding for Shiga toxins

A dual promoter system regulating λ DNA replication initiation

Transcription and DNA replication are tightly regulated to ensure coordination of gene expression with growth conditions and faithful transmission of genetic material to progeny. A large body of evidence has accumulated, indicating that encounters between protein machineries carrying out DNA and RNA synthesis occur in vivo and may have important regulatory consequences. This feature may be exacerbated in the case of compact genomes, like the one of bacteriophage λ, used in our study. Transcription that starts at the rightward pR promoter and proceeds through the λ origin of replication and downstream of it was proven to stimulate the initiation of λ DNA replication. Here, we demonstrate that the activity of a convergently oriented pO promoter decreases the efficiency of transcription starting from pR. Our results show, however, that a lack of the functional pO promoter negatively influences λ phage and λ-derived plasmid replication. We present data, suggesting that this effect is evoked by the enhanced level of the pR-driven transcription, occurring in the presence of the defective pO, which may result in the impeded formation of the replication initiation complex. Our data suggest that the cross talk between the two promoters regulates λ DNA replication and coordinates transcription and replication processes

Wprowadzenie

Wprowadzenie do nr 270 Acta Universitatis Lodziensis, Folia Oeconomic