Determination of the transcription initiation site and identification of the protein product of the regulatory gene xylR for xyl operons on the TOL plasmid

Abstract

The xylR gene is a regulatory gene on the TOL plasmid, which acts in a positive manner on xyl operons for degradation of toluene and xylenes in Pseudomonas putida. A DNA fragment containing the xylR promoter region was cloned on promoter-probing vectors, and its nucleotide sequence was determined. The transcription initiation site of the xylR gene was determined in cells of P. putida and Escherichia coli by S1 nuclease and reverse transcriptase mapping. Two initiation sites were detected which were identical in both P. putida and E. coli. The amounts of mRNA synthesized in both bacterial cells were almost the same and independent of the inducers for xyl operons. The consensus sequences for E. coli promoters were found in the region preceding the respective transcription initiation sites. The product of the xylR gene was identified by the maxicell system as a protein with an approximate molecular weight of 67,000.