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J. Biol. Chem., Vol. 279, Issue 19, 19860-19866, May 7, 2004

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Direct Stimulation of the paQ Promoter by the Transcription Effector Guanosine-3',5'-(bis)pyrophosphate in a Defined in Vitro System^*

Katarzyna Potrykus, Grzegorz Wegrzyn¶, and V. James Hernandez||**

From the Department of Molecular Biology, University of Gdansk, Kladki 24, 80-822 Gdansk, Poland and the ^||Department of Microbiology, State University of New York at Buffalo, Buffalo, New York, 14214

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 paQ 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.

Received for publication, December 8, 2003 , and in revised form, February 27, 2004.

^* This work was supported by National Institutes of Health Grants GM57189 and TW01244 and Polish Ministry of Scientific Research and Information Technology Grant 3P04A 049 24.

Supported by a Young Scientist stipend from the Foundation for Polish Science and a grant from the Foundation for the Development of Gdansk University.

^¶ Supported by Foundation for Polish Science Subsidy Grant 14/2000.

^** To whom correspondence should be addressed. Tel.: 716-829-2141; Fax: 716-829-2158; E-mail: vjh{at}buffalo.edu.

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