ABSTRACT
DNA primase is an enzyme required for replication of both chromosomes and vast majority of plasmids. Guanosine tetra- and penta-phosphate (ppGpp and pppGpp, respectively) are alarmones of the bacterial stringent response to starvation and stress conditions, and act by modulation of the RNA polymerase activity. Recent studies indicated that the primase-catalyzed reaction is also inhibited by (p)ppGpp in Bacillus subtilis, where a specific regulation of DNA replication elongation, the replication fork arrest, was discovered. Although in Escherichia coli such a replication regulation was not reported to date, here we show that E. coli DnaG primase is directly inhibited by ppGpp and pppGpp. However, contrary to the B. subtilis primase response to the stringent control alarmones, the E, coli DnaG was inhibited more efficiently by ppGpp than by pppGpp.
Subject(s)
Endodeoxyribonucleases/antagonists & inhibitors , Escherichia coli Proteins/antagonists & inhibitors , Escherichia coli/enzymology , Exodeoxyribonucleases/antagonists & inhibitors , Guanosine Tetraphosphate/pharmacology , Amino Acid Sequence , Bacillus subtilis/enzymology , DNA Primase , DNA Primers/metabolism , DnaB Helicases/metabolism , Endodeoxyribonucleases/chemistry , Enzyme Inhibitors/pharmacology , Escherichia coli/drug effects , Escherichia coli Proteins/chemistry , Exodeoxyribonucleases/chemistry , Guanosine Diphosphate/pharmacology , Guanosine Pentaphosphate/pharmacology , Models, Molecular , Molecular Sequence Data , Sequence Homology, Amino AcidABSTRACT
The stringent response effector, guanosine tetraphosphate (ppGpp), adjust gene expression and physiology in bacteria, by affecting the activity of various promoters. RNA polymerase-interacting protein, DksA, was proposed to be the co-factor of ppGpp effects; however, there are reports suggesting independent roles of these regulators. Bacteriophage lambda major lytic promoter, pR, is down-regulated by the stringent response and ppGpp. Here, we present evidence that DksA significantly stimulates pR-initiated transcription in vitro in the reconstituted system. DksA is also indispensable for pR activity in vivo. DksA-mediated activation of pR-initiated transcription is predominant over ppGpp effects in the presence of both regulators in vitro. The possible role of the opposite regulation by ppGpp and DksA in lambda phage development is discussed. The major mechanism of DksA-mediated activation of transcription from pR involves facilitating of RNA polymerase binding to the promoter region, which results in more productive transcription initiation. Thus, our results provide evidence for the first promoter inhibited by ppGpp that can be stimulated by the DksA protein both in vivo and in vitro. Therefore, DksA role could be not only independent but antagonistic to ppGpp in transcription regulation.
Subject(s)
Bacteriophage lambda/genetics , Escherichia coli Proteins/metabolism , Guanosine Tetraphosphate/metabolism , Promoter Regions, Genetic , Transcription, Genetic , DNA/metabolism , Transcriptional ActivationABSTRACT
Escherichia coli Integration Host Factor (IHF) regulates transcription from some bacterial and phage promoters by affecting DNA topology. Here we demonstrate that IHF affects transcription from bacteriophage lambdapR promoter and the ptac promoter located on plasmids that contain IHF-binding sites. The IHF consensus sites are abundant and they can bind the IHF protein as shown in in vitro studies. The SeqA protein has a role in the complex regulation of pR activity, together with other factors altering DNA topology. Down-regulation of the transcription from ptac in the absence of IHF, together with IHF- and SeqA-mediated effects on pR, suggest that DNA topology cannot be underestimated when assessing in vivo promoters' activity. This may have a significant impact on experiments employing recombinant genes cloned in plasmids and on choosing appropriate plasmid vectors.
