ORIT-PROCESSING AND REGULATORY ROLES OF TRWA PROTEIN IN PLASMID R388 CONJUGATION

TrwA protein was purified from an overproducing Esherichia coil strain and characterized as a 53 kDa tetrameric DNA-binding protein. Gel shi ft assays showed that TrwA bound specifically to the oriT sequence of plasmid R388. DNAse I footprinting analysis defined two DNA regions wi thin oriT (sites A and B) that were protected by TrwA. At low TrwA con centrations only region A was protected (K-D = 4 x 10(-8)M) while regi on B required higher TrwA concentrations (K-D = 4 x 10(-7) M). As a re sult of its binding to oriT, TrwA was found to perform two biochemical activities related to its role in R388 conjugation. First, TrwA bindi ng to oriT resulted in transcriptional repression of the traABC operon as indicated by its effect on the beta-galactosidase activity of tran scriptional fusions in trwB and trwC, and by direct measurement of the trwA mRNA levels by hybridization. This result was further confirmed by the fact that TrwA overexpression resulted in lowered conjugation f requencies. Second, TrwA enhanced the relaxation activity of TrwC in v itro. This effect was correlated to a 10(5)-fold increase in the frequ ency of conjugation in vivo and was shown to be independent of the reg ulation of transcription. Thus, TrwA shows functional similarities to protein TraY of F-like plasmids, that could be correlated to a structu ral similarity In their DNA-binding motifs. (C) 1997 Academic Press Li mited.