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Stimulation of the p_R promoter by Escherichia coli SeqA protein requires downstream GATC sequences and involves late stages of transcription initiation

Robert ye¹

Grzegorz Wgrzyn¹

Alicja Wgrzyn²

Agnieszka Szalewska-Paasz¹

¹ Department of Molecular Biology, University of Gdansk, Kladki 24, 80-822 Gdansk, Poland
² Laboratory of Molecular Biology (affiliated with University of Gdansk), Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Kladki 24, 80-822 Gdansk, Poland

Correspondence
Agnieszka Szalewska-Paasz
szalewsk{at}biotech.univ.gda.pl

Escherichia coli SeqA protein is a major negative regulator of chromosomal DNA replication acting by sequestration, and thus inactivation, of newly formed oriC regions. However, other activities of this protein have been discovered recently, one of which is regulation of transcription. SeqA has been demonstrated to be a specific transcription factor acting at bacteriophage promoters p_I, p_aQ and p_R. While SeqA-mediated stimulation of p_I and p_aQ occurs by facilitating functions of another transcription activator protein, cII, a mechanism for stimulation of p_R remains largely unknown. Here, it has been demonstrated that two GATC sequences, located 82 and 105 bp downstream of the p_R transcription start site, are necessary for this stimulation both in vivo and in vitro. SeqA-mediated activation of p_R was as effective on a linear DNA template as on a supercoiled one, indicating that alterations in DNA topology are not likely to facilitate the SeqA effect. In vitro transcription analysis demonstrated that the most important regulatory effect of SeqA in p_R transcription occurs after open complex formation, namely during promoter clearance. SeqA did not influence the appearance and level of abortive transcripts or the pausing during transcription elongation. Interestingly, SeqA is one of few known prokaryotic transcription factors which bind downstream of the regulated promoter and still act as transcription activators.