Direct Stimulation of the λpaQ Promoter by the Transcription Effector Guanosine-3′,5′-(bis)pyrophosphate in a Defined in Vitro System

Katarzyna Potrykus,Grzegorz Wegrzyn,V James Hernandez

doi:10.1074/jbc.m313378200

Katarzyna Potrykus, Grzegorz Wegrzyn + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m313378200

Copy DOI

Abstract

The bacterial response to nutritional deprivation, called the stringent response, results in the introduction of the specific nucleotide guanosine-3',5'-(bis) pyrophosphate (ppGpp). This nucleotide interacts with RNA polymerase and alters its action so that transcription from certain promoters is inhibited, whereas transcription from others seems to be activated. The exact mechanism of transcriptional stimulation by ppGpp in vivo remains unknown. A passive control model has been proposed according to which transcription inhibition during the stringent response at several very active promoters, like those for rRNA and tRNA genes, makes more free RNA polymerase (RNAP) molecules available for transcription at promoters with weak binding affinities for RNAP, thus leading to their passive activation. Among promoters whose transcription is activated by ppGpp in vivo is the histidine operon promoter (hisGp). However, in vitro it is only possible to demonstrate this effect in a coupled transcription-translation system. Here we demonstrate, using another in vivo ppGpp-stimulated promoter, the phage lambdapaQ promoter, that activation by ppGpp in a defined in vitro system is direct. A systematic study of ppGpp effects on the stimulation of paQ revealed that, as in the case of promoters inhibited by this nucleotide, ppGpp decreases the half-life of paQ open complexes. Our results also indicate that the equilibrium binding affinity of RNA polymerase to paQ seems not to be affected in the presence of ppGpp. Our data indicate that the mechanism underlying ppGpp stimulation of paQ is due to an increased rate of productive open complex formation.

Highlights

When exposed to nutritional deprivation, bacteria respond quickly and efficiently to changing environmental conditions
A systematic study of ppGpp effects on the stimulation of paQ revealed that, as in the case of promoters inhibited by this nucleotide, ppGpp decreases the half-life of paQ open complexes
In this study we investigated in vitro the phage ␭paQ promoter, a promoter previously uncharacterized for ppGpp stimulation in vitro, but which was previously shown to be stimulated by ppGpp in vivo [9]

Summary

Introduction

When exposed to nutritional deprivation, bacteria respond quickly and efficiently to changing environmental conditions. To shut down protein synthesis, stable RNA synthesis (i.e. rRNA and tRNA) is inhibited Such action is mediated by a small transcription effector molecule, guanosine-3Ј,5Ј-(bis)pyrophosphate (ppGpp),. In contrast to the passive model, it was shown by Choy [7] that, in a mixed template in vitro transcription system containing both positively and negatively controlled promoters, transcriptional inhibition and stimulation mediated by ppGpp are independent of each other, supporting a direct and active mode of positive transcription control by ppGpp. Still, the conclusion of Choy was based on studies employing an S-30 cell extract and, whether ppGpp interacts directly with RNA polymerase to stimulate transcription or whether another factor participates in this process remained unknown. Our results clearly demonstrate that effect of ppGpp on ␭paQ is direct, and, this promoter’s activation in vivo cannot be attributed solely to passive control and does not necessarily require any additional cellular factor

Objectives

Results

Conclusion