Regulatory mechanisms in expression of the traY-I operon of sex factor plasmid R100: involvement of traJ and traY gene products.

BACKGROUND

The plasmid R100 encodes tra genes essential for conjugal DNA transfer in Escherichia coli. Genetic evidence suggests that the traJ gene encodes a positive regulator for the traY-I operon, which includes almost all the tra genes located downstream of traJ. The molecular mechanism of regulation by TraJ, however, is not yet understood. traY is the most proximal gene in the traY-I operon. TraY promotes DNA transfer by binding to a site, sbyA, near the origin of transfer. TraY is suggested to have another role in regulation of the traY-I operon, since it binds to two other sites, named sbyB and sbyC, located in the region preceding traY-I.

RESULTS

Using a traY-lacZ fusion gene, we showed that the traY-I operon was expressed only in the presence of traJ. The TraJ-dependent expression of traY-I required the E. coli arcA gene, which encodes a host factor required for conjugation. TraJ-dependent transcription occurred from a promoter (named pY) located upstream of traY-I. The isolated TraJ protein was found to bind to a dyad symmetry sequence, named sbj (specific binding site of TraJ), which existed in the intergenic region between traJ and traY-I. We also demonstrated that TraY repressed the TraJ-dependent expression of traY-I at the TraY binding sites, sbyB and sbyC, which overlapped with pY.

CONCLUSIONS

TraJ is a protein which binds to the sbj site in the region upstream of the promoter pY and positively regulates expression of the traY-I operon in the presence of the E. coli arcA gene. Since sbj is located 93bp upstream of pY in the intergenic region between traJ and traY-I, TraJ presumably contacts with a transcription apparatus to promote transcription from pY. TraY, which is known to activate the initiation of conjugal DNA transfer, has a new role in the transcriptional autoregulation of traY-I expression. At levels which are sufficient to initiate conjugal DNA transfer, TraY represses traY-I transcription in the presence of TraJ.