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 ␤-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 (6thio-ppGpp), was used to localize the ppGpp binding site. In in vitro

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... cription 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 crosslinker. Cross-linking of RNA polymerase was directed primarily to the ␤-subunit and could be competed efficiently by native ppGpp but not by GTP or GDP. Cyanogen bromide digestion analysis of the cross-linked ␤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 ␤-subunit and a C-terminal fragment of the ␤-subunit. We propose that the N terminus of ␤ together with the C terminus of ␤ constitute a modular ppGpp binding site. 1 The abbreviations used are: RNAP, RNA polymerase; mAb, monoclonal antibody; MALDI-TOF MS, matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry.