Binding of the transcription effector ppGpp to Escherichia coli RNA polymerase is allosteric, modular, and occurs near the N terminus of the beta'-subunit.
Among the prokaryotae, the nucleotide ppGpp is a second messenger of physiological stress and starvation. The target of ppGpp is RNA polymerase, where it putatively binds and alters the enzyme's activity. Previous data had implicated the beta-subunit of Escherichia coli RNA polymerase as containing a single ppGpp binding site. In this study, a photocross-linkable derivative of ppGpp, 6-thioguanosine-3',5'-(bis)pyrophosphate (6-thio-ppGpp), was used to localize the ppGpp binding site. In in vitro transcription assays, 6-thio-ppGpp inhibited transcription from the argT promoter identically to bona fide ppGpp. The thio group of 6-thio-ppGpp is directly photoactivatable and is thus a zero-length cross-linker. Cross-linking of RNA polymerase was directed primarily to the beta'-subunit and could be competed efficiently by native ppGpp but not by GTP or GDP. Cyanogen bromide digestion analysis of the cross-linked beta'-subunit was consistent with an extreme N-terminal cross-link. To assess allosteric consequences of ppGpp binding to RNA polymerase, high level trypsin resistance in the presence and absence of ppGpp was monitored. Trypsin digestion of RNA polymerase bound to ppGpp leads to protection of an N-terminal fragment of the beta'-subunit and a C-terminal fragment of the beta-subunit. We propose that the N terminus of beta' together with the C terminus of beta constitute a modular ppGpp binding site.
Toulokhonov
I I
II
Department of Microbiology, Center of Microbial Pathogenesis, State University of New York at Buffalo School of Medicine, Buffalo, New York 14214, USA.
Shulgina
I
I
Hernandez
V J
VJ
D000494:Allosteric Regulation; D000495:Allosteric Site; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D006153:Guanosine Diphosphate / Q000494:pharmacology; D006159:Guanosine Tetraphosphate / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000378:metabolism* / Q000494:pharmacology; D007700:Kinetics; D010446:Peptide Fragments / Q000737:chemistry; D011401:Promoter Regions, Genetic; D021122:Protein Subunits; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013873:Thionucleotides / Q000138:chemical synthesis / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects*
C422993:6-thioguanosine-3',5'-(bis)pyrophosphate; D010446:Peptide Fragments; D021122:Protein Subunits; D013873:Thionucleotides; D006153:Guanosine Diphosphate; D006159:Guanosine Tetraphosphate; D012321:DNA-Directed RNA Polymerases