The differential effects of guanosine tetraphosphate on open complex formation at the Escherichia coli ribosomal protein promoters rplJ and rpsA P1

Abstract

The effects of guanosine tetraphosphate (ppGpp) on inhibition of single-round in vitro transcription and on the kinetics of open complex formation were investigated at the Escherichia coli ribosomal protein promoters rplJ and rpsA P1. The two promoters differ in their saturation characteristics and sensitivities to ppGpp. With a 10:1 molar ratio of RNA polymerase (RNAP) to DNA, saturation of transcription activity and weak inhibition (≈ 30%) are observed at rplJ, in contrast to the weak activity and strong inhibition (≈ 80%) at rpsA P1. In the absence of ppGpp, the two promoters show a threefold difference in the overall rate constants of association (k_a) (6.5× 10⁷ M^-1 s^-1 at rplJ and 2.0× 10⁷ M^-1 s^-1 at rpsA P1), while the dissociation rate constants (k_d) are similar (≈ 4.8× 10^-5 s^-1). The addition of ppGpp causes a twofold reduction in k₂ (isomerisation constant) at rplJ and a threefold decrease in K_B (equilibrium constant of RNAP binding) at rpsA P1. There is a significant twofold increase in k_d at rplJ, compared with smaller changes at rpsA P1 and at the non-stringent lacUV5 promoter. These results indicate that ppGpp affects the formation and stability of the open complex at the rplJ promoter, in contrast to the inhibition of RNAP binding to the rpsA P1 promoter.