DIFFERENTIAL INTERACTION OF THE TRANSCRIPTION FACTOR PRFA AND THE PRFA-ACTIVATING FACTOR (PAF) OF LISTERIA-MONOCYTOGENES WITH TARGET SEQUENCES

The interaction of the purified PrfA transcription factor with the reg ulatory sequences located upstream of the PrfA-dependent listeriolysin (hly) and internalin (inlA) genes was studied in the presence and in the absence of Paf (PrfA-activating factor)-containing extracts. It is shown that PrfA protein is able to bind, independently of additional factors, to a 109 bp DNA fragment including the entire hly promoter se quence with the anticipated PrfA binding site ('PrfA-box'). PrfA alone , but not in combination with Paf, can also bind to a shorter target s equence of 28 bp comprising essentially the PrfA-box of the hly promot er. The addition of a Paf-containing extract does not lead to signific ant protein binding to these two hly target sequences in the absence o f PrfA but converts the complex (CIII) consisting of PrfA and the 109 bp hly DNA fragment to a slower migrating PrfA-Paf-DNA complex (CI). I ncubation of cell-free extracts of wildtype Listeria monocytogenes wit h the 109 bp DNA fragment leads to the formation of CI. The addition o f polyclonal PrfA antibodies causes a supershift of CIII. Purified Prf A and PrfA-Paf also bind to a DNA fragment containing the PrfA-depende nt promoter P2 of inlA, albeit at a lower rate when compared with the corresponding hly sequence. In contrast to the hly target DNA, the inl A promoter sequence efficiently binds Paf alone, and this Paf binding reduces that of PrfA and PrfA-Paf to the inlA target DNA. DNase I foot print experiments show that purified PrfA protects sequences of dyad s ymmetry previously proposed as PrfA binding sites in the hly and in th e inlA promoter regions.