Analysis of the MRNA Structure of the Pseudomonas Putida TOL Meta Fission Pathway Operon Around the Transcription Initiation Point, the XylTE and the XylFJ Regions

The 13 genes encoded by the meta-cleavage operon (approx. 11 kb) of Pseudomonas putida TOL plasmid pWW0 are transcribed from a single promoter, Pm. In P. putida, transcription from Pm was strictly dependent on the presence of effector-activated XylS protein. Three regions of the transcript were analyzed in the wild-type strain of P. putida (pWW0) by S1 nuclease protection and primer extension analyses. A major point of transcription initiation was found in the most 5'-end of the operon, which defined a -10/-35 promoter. Another main mRNA 5'-end point located within the coding sequence of the first gene of the operon was detected and identified as a stable endonucleolytic cleavage product, which rendered this gene inactive for further translation. The estimated half-life of this leader region was 2 min. Analyses of the transcripts at xylLTE and xylFJ regions revealed the presence of several relatively stable mRNA products with 5'-ends located along these regions, which arose from specific endonucleolytic mRNA cleavage. The degradation of the stable intermediate products observed after the addition of rifampicin was similar for all the products, presenting a delay of approx. 10 min, followed by an exponential decay, with a half-life that ranged between 6 and 10 min.