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 regulatory 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 109bp DNA fragment including the entire hly promoter sequence with the anticipated PrfA binding site ('PrfA-box'). PrfA alone, but not in combination with Paf, can also bind to a shorter target sequence of 28 bp comprising essentially the PrfA-box of the hly promoter. The addition of a Paf-containing extract does not lead to significant protein binding to these two hly target sequences in the absence of 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). Incubation of cell-free extracts of wild-type Listeria monocytogenes with the 109 bp DNA fragment leads to the formation of CI. The addition of polyclonal PrfA antibodies causes a supershift of CIII. Purified PrfA and PrfA-Paf also bind to a DNA fragment containing the PrfA-dependent promoter P2 of inlA, albeit at a lower rate when compared with the corresponding hly sequence. In contrast to the hly target DNA, the inlA promoter sequence efficiently binds Paf alone, and this Paf binding reduces that of PrfA and PrfA-Paf to the inlA target DNA. DNase I footprint experiments show that purified PrfA protects sequences of dyad symmetry previously proposed as PrfA binding sites in the hly and in the inlA promoter regions.
Dickneite
C
C
Theodor-Boveri-Institut für Biowissenschaften der Universität Würzburg (Lehrstuhl für Mikrobiologie), Germany.
Böckmann
R
R
Spory
A
A
Goebel
W
W
Sokolovic
Z
Z
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001427:Bacterial Toxins; D001665:Binding Sites; D002499:Centrifugation, Density Gradient; D015342:DNA Probes; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D006360:Heat-Shock Proteins / Q000235:genetics; D006460:Hemolysin Proteins / Q000235:genetics; D015151:Immunoblotting; D008089:Listeria monocytogenes / Q000235:genetics* / Q000472:pathogenicity; D010454:Peptide Termination Factors; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012045:Regulatory Sequences, Nucleic Acid; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics; D014774:Virulence / Q000235:genetics
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D015342:DNA Probes; D004269:DNA, Bacterial; D006360:Heat-Shock Proteins; D006460:Hemolysin Proteins; D010454:Peptide Termination Factors; D015534:Trans-Activators; D014157:Transcription Factors; C069761:internalin protein, Bacteria; C038705:hlyA protein, Listeria monocytogenes