title: 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.
abstract: 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.
authors.affiliation: Biocenter (Microbiology), University of Würzburg, 97074 Würzburg, Germany.
authors.forename: Qin
authors.initials: Q
authors.lastname: Luo
authors.forename: Marcus
authors.initials: M
authors.lastname: Rauch
authors.forename: Alexandra K
authors.initials: AK
authors.lastname: Marr
authors.forename: Stefanie
authors.initials: S
authors.lastname: Müller-Altrock
authors.forename: Werner
authors.initials: W
authors.lastname: Goebel
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000502:physiology*; D001427:Bacterial Toxins / Q000235:genetics; D001483:Base Sequence; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D006160:Guanosine Triphosphate / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000235:genetics / Q000502:physiology; D006460:Hemolysin Proteins; D008089:Listeria monocytogenes / Q000235:genetics* / Q000472:pathogenicity / Q000502:physiology; D008565:Membrane Proteins / Q000235:genetics / Q000502:physiology; D008666:Metalloendopeptidases / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010454:Peptide Termination Factors; D011401:Promoter Regions, Genetic; D017384:Sequence Deletion; D015534:Trans-Activators / Q000502:physiology*; D014157:Transcription Factors / Q000502:physiology; D024363:Transcription Initiation Site; D014158:Transcription, Genetic; D037521:Virulence Factors / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D006360:Heat-Shock Proteins; D006460:Hemolysin Proteins; D008565:Membrane Proteins; D010454:Peptide Termination Factors; D015534:Trans-Activators; D014157:Transcription Factors; D037521:Virulence Factors; C103178:inlC protein, Listeria monocytogenes; C072980:actA protein, Listeria monocytogenes; D006160:Guanosine Triphosphate; D008666:Metalloendopeptidases; C080367:mpl protein, Listeria monocytogenes; C038705:hlyA protein, Listeria monocytogenes