The Mechanism of Regulation of Bacteriophage λ pR Promoter Activity by Escherichia coli DnaA Protein

Abstract

Apart from its function as an initiator of DNA replication, the Escherichia coli DnaA protein is also a specific transcription factor. It activates and represses a number of promoters. However, mechanisms of transcription stimulation by DnaA remained unknown. Bacteriophage p R promoter is one of the promoters activated by DnaA. It was reported previously that DnaA binds downstream of the p R promoter and perhaps interacts with the RNA polymerase ␤ subunit. Here we demonstrate that DnaA positively regulates transcription from p R by stimulation of two steps in transcription initiation: RNA polymerase binding to the promoter region and promoter escape. For this transcription activation, two weak DnaA boxes located downstream of p R are necessary and sufficient. Such a mechanism of transcription activation and location of the activator-binding sites relative to the transcription start point are unusual in prokaryotes. Changes in the distance between the transcription start point and the first DnaA box by 5 and 10 bp and alterations in the orientation of these boxes did not abolish the stimulation of transcription by DnaA, but the efficiency of the promoter activation was different for various mutations. It seems plausible that formation of higher order nucleoprotein structures, involving DNA looping, is necessary for effective stimulation of the p R promoter. At high concentrations, DnaA is a repressor of p R rather than an activator. This repression was found to be because of inhibition of RNA polymerase binding to the promoter region.

A transcriptional response to replication status mediated by the conserved bacterial replication protein DnaA

“…There are examples in E. coli where one site with one mismatch and another more degenerate site are sufficient for DnaA-dependent regulation. Additionally, some genes controlled by DnaA have binding sites downstream of the promoter regions, sometimes within an ORF (47,48). These cases would have been missed by our analysis.…”

Modulation of λ plasmid and phage DNA replication by Escherichia coli SeqA protein

“…The mechanism of this phenomenon remains to be elucidated; nevertheless, it is worth noting that SeqA binds to a region of the l p R promoter and regulates its activity (S omiń ska et al, 2001), and that a decrease in the abundance of the stable O protein fraction is also observed in cells bearing a derivative of l plasmid (pTCl2) in which p R is replaced by another promoter (p tet ) (HermanAntosiewicz et al, 1998a) [the activity of the p tet promoter has been demonstrated to be SeqA independent (S omiń ska et al, 2001)]. Moreover, another regulator of p R activity, the DnaA protein (Szalewska-Pa asz et al, 1998;Glinkowska et al, 2003), has also been suggested to be a factor that influences the stability of the l replication complex (Herman-Antosiewicz et al, 1998a), and an interplay between DnaA and SeqA at the p R promoter region has been clearly demonstrated (S omiń ska et al, 2003b). In this light, seqA dysfunction-mediated suppression of the effects of certain dnaA alleles on the transformation of E. coli cells with l plasmids, demonstrated previously (Glinkowska et al, 2001), and on the directionality of l plasmid replication, suggested in this report, may be important to understand the regulation of l DNA replication initiation.…”

“…Examples of studies of the possible role of SeqA in the regulation of replication initiated from origins different from oriC include relatively preliminary observations only, namely the demonstration of the binding of this protein to bacteriophage P1 ori (Brendler et al, 1995), and the findings that the copy number of plasmids derived from bacteriophage l is decreased in a seqA mutant host (S omiń ska et al, 2001) and that the DseqA mutation can suppress incompatibility between l plasmids and certain dnaA(ts) mutants (S omiń ska et al, 2003b;Glinkowska et al, 2003). Here, we investigated the role of SeqA in the regulation of l DNA replication in more detail.…”

“…Although the use of products of cellular genes by viruses may sometimes appear unusual, studies of such processes have often led to the discovery of important properties of host proteins. This applies also to bacteriophages and their hosts, including one of the most intensively studied models in molecular biology, phage l. Examples of the processes described above are the use of E. coli LamB protein, normally allowing uptake of maltose and maltodextrins, for phage adsorption on the cell surface (Vinga et al, 2006), employment of heat-shock proteins in l DNA replication and morphogenesis (Polissi et al, 1995; Taylor & Węgrzyn, 1995), and involvement of the replication initiator protein DnaA in the regulation of transcription from the l p R promoter (Szalewska-Pa asz et al, 1998;Glinkowska et al, 2003). Therefore, we aimed to investigate the role of SeqA in the control of replication of the bacteriophage l genome and plasmids derived from this phage.…”

FleQ DNA Binding Consensus Sequence Revealed by Studies of FleQ-Dependent Regulation of Biofilm Gene Expression in Pseudomonas aeruginosa

“…A few transcription activator proteins have been shown to bind downstream of the transcription start site, but little is known concerning their mechanism of activation (32)(33)(34). In the case of regulation of bacteriophage lambda pR promoter activity by DnaA, the location of the binding site downstream of the transcription start site allows a direct interaction between DnaA and the RNA polymerase ␤ subunit (32). Weak binding of DnaA to DNA may contribute to removal of this activator after transcription initiation (32).…”