Guanosine Tetraphosphate Has a Similar Affinity for Each of Its Two Binding Sites on Escherichia coli RNA Polymerase

During nutrient deprivation, the bacterial cell undergoes a stress response known as the stringent response. This response is characterized by induction of the nucleotide derivative guanosine tetraphosphate (ppGpp) that dramatically modulates the cell's transcriptome. In Escherichia coli, ppGpp regulates transcription of as many as 750 genes within 5 min of induction by binding directly to RNA polymerase (RNAP) at two sites similar to 60 angstrom apart. One proposal for the presence of two sites is that they have different affinities for ppGpp, expanding the dynamic range over which ppGpp acts. We show here, primarily using the Differential Radial Capillary Action of Ligand Assay (DRaCALA), that ppGpp has a similar affinity for each site, contradicting the proposal. Because the ppGpp binding sites are formed by interactions of the beta' subunit of RNAP with two small protein factors, the omega subunit of RNAP which contributes to Site 1 and the transcription factor DksA which contributes to Site 2, variation in the concentrations of omega or DksA potentially could differentially regulate ppGpp occupancy of the two sites. It was shown previously that DksA varies little at different growth rates or growth phases, but little is known about variation of the omega concentration. Therefore, we raised an anti-omega antibody and performed Western blots at different times in growth and during a stringent response. We show here that omega, like DksA, changes little with growth conditions. Together, our data suggest that the two ppGpp binding sites fill in parallel, and occupancy with changing nutritional conditions is determined by variation in the ppGpp concentration, not by variation in omega or DksA.