ABSTRACT
Analysis of the sequence immediate upstream of ohrrevealed an open reading frame, designated ohrR, with the potential to encode a 17-kDa peptide with moderate amino acid sequence homology to the MarR family of negative regulators of gene expression. ohrR was transcribed as bicistronic mRNA with ohr, while ohr mRNA was found to be 95% monocistronic and 5% bicistronic with ohrR. Expression of both genes was induced by tert-butyl hydroperoxide (tBOOH) treatment. High-level expression ofohrR negatively regulated ohr expression. This repression could be overcome by tBOOH treatment. In vivo promoter analysis showed that the ohrR promoter (P1) has organic peroxide-inducible, strong activity, while the ohrpromoter (P2) has constitutive, weak activity. Only P1 is autoregulated by OhrR. ohr primer extension results revealed three major primer extension products corresponding to the 5′ ends ofohr mRNA, and their levels were strongly induced by tBOOH treatment. Sequence analysis of regions upstream of these sites showed no typical Xanthomonas promoter. Instead, the regions can form a stem-loop secondary structure with the 5′ ends ofohr mRNA located in the loop section. The secondary structure resembles the structure recognized and processed by RNase III enzyme. These findings suggest that the P1 promoter is responsible for tBOOH-induced expression of the ohrR-ohr operon. The bicistronic mRNA is then processed by RNase III-like enzymes to give high levels of ohr mRNA, while ohrR mRNA is rapidly degraded.
Determination of the Secondary Structure of an RNA fragment in Solution: Selective 2`-Hydroxyl Acylation Analyzed by Primer Extension Assay (SHAPE)
