Most known virulence genes of Listeria monocytogenes are regulated by the transcriptional factor PrfA. Using our recently established In vitro transcription system, we have studied the PrfA-dependent promoter (PinlC) regulating the expression of the small, secreted internalin C. PrfA-dependent and PrfA-independent transcription is observed starting from PinlC in vitro and in vivo, suggesting the presence of two apparently overlapping promoters both of which use the same -10 box. Although the PrfA-dependent transcription requires, as expected, the PrfA-box, PrfA-independent transcription depends on a -35 box located directly downstream of the PrfA-box. PrfA-independent transcription starts at A, 7 bp down-stream of the common -10 box (A7), and Is strongly inhibited by PrfA because of the close proximity of the PrfA binding site to the -35 box. PrfA-dependent transcription starts preferentially at G5 but, in the absence of this start nucleotide, alternative start sites at A positions 7 or 8 bp downstream of the -10 box can also be used. The -35 box of the PrfA-independent promoter can be functionally inactivated without affecting PrfA-dependent transcription as long as the distance between the PrfA-box and the -10 box remains fixed to 22 (or 23) bp. Vice versa, the PrfA-box can be deleted without affecting PrfA-independent transcription from PinlC, which is no longer inhibited by PrfA. The PrfA-dependent transcription initiation needs, in contrast to the PrfA-independent one, the presence of a high concentration of GTP (and ATP) but not of CTP and UTP. Overlapping PrfA-dependent and PrfA-independent promoter activity was also demonstrated for the mpl promoter (Pmpl). Again, PrfA-dependent transcription starting at Pmpl is dominant at high GTP concentration and PrfA-independent transcription at low GTP. Here too, the PrfA-dependent and the PrfA-independent promoters share the same -10 box characteristic of SigA-loaded RNA polymerase. High GTP concentration also appears to be necessary for transcription initiation at other PrfA-dependent promoters (Phly, PactA) but not at the PrfA-independent promoter PinlC-m8.
ASJC Scopus subject areas
- Molecular Biology