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Journal of Bacteriology, August 1999, p. 4704-4707, Vol. 181, No. 15
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Involvement of the RNA Polymerase alpha -Subunit C-Terminal Domain in LuxR-Dependent Activation of the Vibrio fischeri Luminescence Genes

Ann M. Stevens,1,* Nobuyuki Fujita,2 Akira Ishihama,2 and E. P. Greenberg3

Department of Biology, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 240611; Department of Molecular Genetics, National Institute of Genetics, Mishima, Shizuoka, 411-8540 Japan2; and Department of Microbiology, University of Iowa, Iowa City, Iowa 522423

Received 19 February 1999/Accepted 24 May 1999

LuxR is a sigma 70 RNA polymerase (RNAP)-dependent transcriptional activator that controls expression of the Vibrio fischeri lux operon in response to an acylhomoserine lactone-cell density signal. We have investigated whether the alpha -subunit C-terminal domain (alpha CTD) of RNAP is required for LuxR activity. A purified signal-independent, LuxR C-terminal domain-containing polypeptide (LuxRDelta N) was used to study the activation of transcription from the luxI promoter in vitro. Initiation of lux operon transcription was observed in the presence of LuxRDelta N and wild-type RNAP but not in the presence of LuxRDelta N and RNAPs with truncated alpha CTDs. We also studied the in vivo role of the RNAP alpha CTD in activation of lux transcription in Escherichia coli. This enabled a comparison of results obtained with full-length LuxR to those obtained with LuxRDelta N. These in vivo studies indicated that both LuxR and LuxRDelta N require the RNAP alpha CTD for activity. The results of DNase I protection studies showed that LuxRDelta N-RNAP complexes can bind and protect the luxI promoter, but with less efficacy when the alpha CTD is truncated in comparison to the wild type. Thus, both in vitro and in vivo experiments demonstrated that LuxR-dependent transcriptional activation of the lux operon involves the RNAP alpha CTD and suggest that alpha CTD-LuxR interactions may play a role in recruitment of RNAP to the luxI promoter.

* Corresponding author. Mailing address: Department of Biology, 4020 Derring Hall, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061. Phone: (540) 231-9378. Fax: (540) 231-9307. E-mail: ams{at}vt.edu.

Journal of Bacteriology, August 1999, p. 4704-4707, Vol. 181, No. 15
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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