ABSTRACT
Pfs expression is required for several metabolic pathways and limits the production of autoinducer-2, a molecule proposed to play a central role in interspecies quorum sensing. The present study reveals physiological conditions and promoter DNA elements that regulate Escherichia coli pfs transcription. Pfs transcription is shown to rely on both sigma 70 and sigma 38 (rpoS), and the latter is subject to induction that increases pfs expression. Transcription is maximal as the cells approach stationary phase, and this level can be increased by salt stress through induction of sigma 38-dependent expression. The pfs promoter is shown to contain both positive and negative elements, which can be used by both forms of RNA polymerase. The negative element is contained within the overlapping dgt promoter, which is involved in purine metabolism. Consideration of the physiological roles of sigma 38 and dgt leads to a model for how autoinducer production is controlled under changing physiological conditions.
Subject(s)
Escherichia coli/physiology , Gene Expression Regulation, Bacterial , Homoserine/analogs & derivatives , N-Glycosyl Hydrolases/genetics , N-Glycosyl Hydrolases/physiology , Purines/metabolism , Transcription, Genetic , Bacterial Proteins/metabolism , DNA-Directed RNA Polymerases/metabolism , Escherichia coli/genetics , Homoserine/biosynthesis , Lactones , N-Glycosyl Hydrolases/biosynthesis , Promoter Regions, Genetic , RNA, Bacterial/analysis , RNA, Bacterial/genetics , RNA, Messenger/analysis , RNA, Messenger/genetics , Sigma Factor/metabolismABSTRACT
During promoter engagement, RNA polymerase must change conformation or isomerize to its active form. These data show that high concentrations of nucleotides assist this isomerization. When binding to fork junction DNA probes is monitored, isomerization can occur without the need for the DNA that overlaps the transcription start site. When the start site is present, nucleoside triphosphates cause polymerase to change conformation in a way that drives cross-linking to the +1 position on the template strand. Preincubation of transcription complexes with 2 mM initiating nucleotide can drive formation of heparin-resistant complexes under conditions in which isomerization is limiting. It is proposed that complete polymerase isomerization can require nucleotide binding, which can assist formation of the active site that engages the transcription start site.
Subject(s)
DNA-Directed RNA Polymerases/chemistry , DNA-Directed RNA Polymerases/genetics , Deoxyribonucleotides/chemistry , Deoxyribonucleotides/genetics , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Base Pairing , Binding Sites/genetics , Catalysis , Cross-Linking Reagents/chemistry , DNA Probes/chemistry , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Hydrogen-Ion Concentration , Isomerism , Nucleic Acid Conformation , Peptide Chain Initiation, Translational/genetics , Protein Conformation , Sigma Factor/chemistry , Sigma Factor/genetics , Templates, Genetic , Transcription Initiation Site , Uridine Triphosphate/chemistryABSTRACT
The rate of transcription of Escherichia coli ribosomal RNA promoters is central to adjusting the cellular growth rate to nutritional conditions. The +1 initiating nucleotide and ppGpp are regulatory effectors of these promoters. The data herein show that in vitro transcription is also regulated by the +2 nucleotide. Both the +1 and +2 nucleotides act by driving polymerase into an altered conformation rather than by increasing the lifetime of transcription complexes. The unique design of the ribosomal promoters may stabilize a distorted state of polymerase that is relieved by the binding of the two nucleotides required for transcription initiation.
Subject(s)
Escherichia coli/metabolism , Nucleotides/chemistry , Promoter Regions, Genetic , Ribosomes/genetics , Ribosomes/physiology , Binding Sites , DNA/chemistry , DNA-Directed RNA Polymerases/chemistry , Dose-Response Relationship, Drug , Gene Expression Regulation, Bacterial , Heparin/pharmacology , Kinetics , Plasmids/metabolism , Protein Conformation , RNA, Ribosomal/chemistry , Transcription, GeneticABSTRACT
23 amino acid substitutions were made in the C7 and C3 regions of pspFDeltaHTH, a protein required to convert sigma(54) closed promoter complexes to open complexes. These mutants were assayed for transcriptional competence, for the ability to hydrolyze ATP, for their multimerization state, and for their ability to interact with sigma(54) and its holoenzyme. C7 region mutants caused the protein to assume a compact form. This property could be mimicked by the addition of ATP, implying that compaction via C7 and ATP is part of the activation process. A number of C3 mutants were important for energy coupling, as indicated previously for several members of this activator family (, ). However, a patch within C3 influenced oligomerization. The C3 region was especially important in interacting with sigma(54) during the transition state but not important in inducing sigma(54) holoenzyme to engage the nontemplate strand of the promoter. It is proposed that both regions contain deterrent functions that prevent premature activation. Overall, the results imply unexpected roles for the C7 and C3 regions of this protein family during promoter activation.
