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J. Biol. Chem., Vol. 278, Issue 47, 47340-47349, November 21, 2003
Fis Stabilizes the Interaction between RNA Polymerase and the Ribosomal Promoter rrnB P1, Leading to Transcriptional Activation*![]() ![]() ![]() From the Laboratory of Molecular Biology, NCI, National Institutes of Health, Bethesda, Maryland 20892 and the ¶Department of Biological Chemistry, David Geffen School of Medicine at UCLA, UCLA, Los Angeles, California 90095-1737 It has been shown that Fis activates transcription of the ribosomal promoter rrnB P1; however, the mechanism by which Fis activates rrnB P1 transcription is not fully understood. Paradoxically, although Fis activates transcription of rrnB P1 in vitro, transcription from the promoter containing Fis sites (as measured from rrnB P1-lacZ fusions) is not reduced in a fis null mutant strain. In this study, we further investigated the mechanism by which Fis activates transcription of the rrnB P1 promoter and the role of Fis in rRNA synthesis and cell growth in Escherichia coli. Like all other stringent promoters investigated so far, open complex of rrnB P1 has been shown to be intrinsically unstable, making open complex stability a potential regulatory step in transcription of this class of promoters. Our results show that Fis acts at this regulatory step by stabilizing the interaction between RNA polymerase and rrnB P1 in the absence of NTPs. Mutational analysis of the Fis protein demonstrates that there is a complete correlation between Fis-mediated transcriptional activation of rrnB P1 and Fis-mediated stabilization of preinitiation complexes of the promoter. Thus, our study indicates that Fis-mediated stabilization of RNA polymerase-rrnB P1 preinitiation complexes, presumably at the open complex step, contributes prominently to transcriptional activation. Furthermore, our in vivo results show that rRNA synthesis from the P1 promoters of several rRNA operons are reduced 2-fold in a fis null mutant compared with the wild type strain, indicating that Fis plays an important role in the establishment of robust rRNA synthesis when E. coli cells are emerging from a growth-arrested phase to a rapid growth phase. Thus, our results resolve an apparent paradox of the role of Fis in vitro and in vivo in the field.
Received for publication, May 23, 2003 , and in revised form, September 12, 2003. * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
|| To whom correspondence should be addressed: Laboratory of Molecular Biology, NCI, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892. Tel.: 301-402-9281; E-mail: djjin{at}helix.nih.gov.
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