IN-VITRO ACTIVITIES OF AN N-TERMINAL TRUNCATED FORM OF XYLR, A SIGMA(54)-DEPENDENT TRANSCRIPTIONAL ACTIVATOR OF PSEUDOMONAS-PUTIDA

A truncated derivative of the XylR protein, which is able to constitut ively activate the sigma(54)-dependent Pu promoter of the TOL (toluene biodegradation) plasmid of Pseudomonas putida, has been purified to h omogeneity and its various activities have been separately examined in vitro. The truncated regulator XylR Delta A was deleted of the signal reception N-terminal module present in wild-type XylR but retained it s central activation domain and the DNA binding segment, located at it s C terminus. XylR Delta A bound to the region -120 to -190 bp upstrea m of the transcription initiation site of the Pu promoter, where previ ous analyses have located the XylR target site. XylR Delta A showed an intrinsic ATPase activity that was strongly stimulated by DNA contain ing the native upstream activation sequences of PEI. Both ATPase activ ity and ATP binding were abolished in mutant G268N in which the Walker A domain of the central module was altered. Mutant R453H lacked ATPas e activity but retained the nucleotide-binding ability of the parental protein. XylR Delta A was able to activate transcription in vitro wit h sigma(54)-RNA polymerase alone, although its activity was enhanced u p to 20-fold in the presence of the integration host factor protein. T he requirements for activation of the Pu promoter in vitro are consist ent with the view that DNA-facilitated oligomerization of the regulato r for an enhanced ATPase activity is the critical event that precedes transcription initiation at sigma(54)-dependent promoters. Furthermore , additional coregulation elements seem to adjust promoter activity in vivo to the physiological status of the cells. (C) 1996 Academic Pres s Limited