Involvement of the RNA polymerase alpha-subunit C-terminal domain in LuxR-dependent activation of the Vibrio fischeri luminescence genes.
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 (alphaCTD) of RNAP is required for LuxR activity. A purified signal-independent, LuxR C-terminal domain-containing polypeptide (LuxRDeltaN) 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 LuxRDeltaN and wild-type RNAP but not in the presence of LuxRDeltaN and RNAPs with truncated alphaCTDs. We also studied the in vivo role of the RNAP alphaCTD in activation of lux transcription in Escherichia coli. This enabled a comparison of results obtained with full-length LuxR to those obtained with LuxRDeltaN. These in vivo studies indicated that both LuxR and LuxRDeltaN require the RNAP alphaCTD for activity. The results of DNase I protection studies showed that LuxRDeltaN-RNAP complexes can bind and protect the luxI promoter, but with less efficacy when the alphaCTD 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 alphaCTD and suggest that alphaCTD-LuxR interactions may play a role in recruitment of RNAP to the luxI promoter.
Stevens
A M
AM
Department of Biology, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061, USA. ams@vt.edu
Fujita
N
N
Ishihama
A
A
Greenberg
E P
EP
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli; D015964:Gene Expression Regulation, Bacterial; D008163:Luminescent Measurements; D009876:Operon; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000378:metabolism*; D015534:Trans-Activators / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D014733:Vibrio / Q000235:genetics* / Q000378:metabolism*
D001426:Bacterial Proteins; C085427:LuxI protein, Bacteria; D012097:Repressor Proteins; D015534:Trans-Activators; D014157:Transcription Factors; C064778:LuxR autoinducer binding proteins; D012321:DNA-Directed RNA Polymerases; C113275:RNA polymerase alpha subunit