Direct stimulation of the lambdapaQ promoter by the transcription effector guanosine-3',5'-(bis)pyrophosphate in a defined in vitro system.
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.
Potrykus
Katarzyna
K
Department of Molecular Biology, University of Gdansk, Kladki 24, 80-822 Gdansk, Poland.
Wegrzyn
Grzegorz
G
Hernandez
V James
VJ
D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D015964:Gene Expression Regulation, Bacterial; D006153:Guanosine Diphosphate / Q000494:pharmacology; D006159:Guanosine Tetraphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D008956:Models, Chemical; D011196:Potassium Permanganate / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D012343:RNA, Transfer / Q000378:metabolism; D013873:Thionucleotides / Q000494:pharmacology; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014764:Viral Proteins
C422993:6-thioguanosine-3',5'-(bis)pyrophosphate; D013873:Thionucleotides; D014157:Transcription Factors; D014764:Viral Proteins; C039000:cII protein, bacteriophage lambda; D011196:Potassium Permanganate; D006153:Guanosine Diphosphate; D006159:Guanosine Tetraphosphate; D012343:RNA, Transfer; D012321:DNA-Directed RNA Polymerases; D003850:Deoxyribonuclease I