In vitro transcription of the Listeria monocytogenes virulence genes inlC and mpl reveals overlapping PrfA-dependent and -independent promoters that are differentially activated by GTP.

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 downstream 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.