A Proximal Promoter Element Required for Positive Transcriptional Control by Guanosine Tetraphosphate and DksA Protein during the Stringent Response

• Published in: The Journal of biological chemistry 2013-07, Vol.288 (29), p.21055-21064
• Main Authors: Gummesson, Bertil, Lovmar, Martin, Nyström, Thomas
• Format: Article
• Language: English
• Subjects: 1961; 1979; 1995; AT Rich Sequence - genetics; ATES OF AMERICA; Bacterial Transcription; Base Composition - genetics; Base Sequence; BOER HA; CELL; CONTROL; Discriminator; DksA; DNA-Directed RNA Polymerases - metabolism; EBBINS CE; ENT GS; Escherichia coli - drug effects; Escherichia coli - genetics; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; ESCHERICHIA-COLI; Gene Expression Regulation, Bacterial; Gene Regulation; Genes, Bacterial - genetics; GROWTH-RATE; Guanosine Tetraphosphate - pharmacology; IN-VIVO; Kinetics; Medical Biotechnology; Medicinsk bioteknologi; Molecular Sequence Data; NATURE; P201; P3292; P636; PPGPP; PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF; Promoter Regions, Genetic; Protein Binding - drug effects; Protein Binding - genetics; RECOGNITION ELEMENT; RIBONUCLEIC-ACID SYNTHESIS; Ribosomal RNA (rRNA); RIBOSOMAL-RNA; RNA Polymerase; SECONDARY CHANNEL; Stress Response; Stringent Control; Transcription, Genetic; UNIVERSAL STRESS-PROTEIN; V105; V17; V373
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M113.479998

The alarmone guanosine tetraphosphate (ppGpp) acts as both a positive and a negative regulator of gene expression in the presence of DksA, but the underlying mechanisms of this differential control are unclear. Here, using uspA hybrid promoters, we show that an AT-rich discriminator region is crucial for positive control by ppGpp/DksA. The AT-rich discriminator makes the RNA polymerase-promoter complex extremely stable and therefore easily saturated with RNA polymerase. A more efficient transcription is achieved when the RNA polymerase-promoter complex is destabilized with ppGpp/DksA. We found that exchanging the AT-rich discriminator of uspA with the GC-rich rrnB-P1 discriminator made the uspA promoter negatively regulated by ppGpp/DksA both in vivo and in vitro. In addition, the GC-rich discriminator destabilized the RNA polymerase-promoter complex, and the effect of ppGpp/DksA on the kinetic properties of the promoter was reversed. We propose that the transcription initiation rate from promoters with GC-rich discriminators, in contrast to the uspA-promoter, is not limited by the stability of the open complex. The findings are discussed in view of models for both direct and indirect effects of ppGpp/DksA on transcriptional trade-offs. Background: ppGpp/DksA regulate genes both positively and negatively during stringency. Results: Positive control by ppGpp/DksA is linked to RNA polymerase-promoter saturation kinetics dependent on the promoter's discriminator. Conclusion: Promoters harboring discriminators that make them easily saturated with RNA polymerase are controlled positively by ppGpp/DksA. Significance: The discriminator is a cis-acting element dictating the response of the promoter to alterations in ppGpp/DksA and RNA polymerase availability.