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J. Biol. Chem., Vol. 276, Issue 2, 1220-1225, January 12, 2001
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From the Department of Microbiology, Center of Microbial
Pathogenesis, State University of New York at Buffalo School of
Medicine, Buffalo, New York 14214
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
Binding of the Transcription Effector ppGpp to Escherichia
coli RNA Polymerase Is Allosteric, Modular, and Occurs Near the N
Terminus of the
'-Subunit*
,
-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 '-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.
*
This work was supported by National Institutes of Health
Grant GM57189.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
On leave from the Limnological Institute of the Russian Academy of
Sciences, Irkutsk, Russia. Current address: Dept. of Bacteriology, University of Wisconsin, Madison, WI 53706.
§
To whom correspondence should be addressed. Tel.: 716-829-2141;
Fax: 716-829-2158; E-mail: vjh@buffalo.edu.
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