RNA polymerase holoenzymes can share a single transcription start site for the Pm promoter. Critical nucleotides in the -7 to -18 region are needed to select between RNA polymerase with sigma38 or sigma32

• Published in: The Journal of biological chemistry 2005-12, Vol.280 (50), p.41315-41323
• Main Authors: Domínguez-Cuevas, Patricia, Marín, Patricia, Ramos, Juan L, Marqués, Silvia
• Format: Article
• Language: English
• Subjects: Bacterial Proteins - chemistry; Bacterial Proteins - metabolism; Base Sequence; beta-Galactosidase - metabolism; Blotting, Western; DNA - chemistry; DNA-Directed RNA Polymerases - chemistry; Escherichia coli - metabolism; Gene Expression Regulation, Bacterial; Heat-Shock Proteins - metabolism; Molecular Sequence Data; Mutation; Nucleotides - chemistry; Oxygen - metabolism; Plasmids - metabolism; Point Mutation; Promoter Regions, Genetic - genetics; Pseudomonas putida - genetics; Pseudomonas putida - metabolism; Sequence Homology, Nucleic Acid; Sigma Factor - metabolism; Transcription Initiation Site; Transcription, Genetic
• ISSN: 0021-9258

The Pm promoter of the benzoate meta-cleavage pathway is transcribed with E sigma32 or E sigma38 according to the growth phase, with an identical transcriptional start site. To investigate sequence determinants in the interaction between either of the two RNA polymerases and Pm, all possible single mutants between positions -7 and -18 were generated, and the activity in the exponential and stationary phases of the resulting mutant promoters was compared. The results precisely delimited a -10 element between positions -7 and -12 (TAGGCT), which defined a promoter sharing nucleotides with both sigma38 and sigma32 consensus. The first two and the last positions of this hexamer were crucial for recognition by both polymerases. Position -10 was the only one specifically recognized by E sigma38, whereas positions -8, -9, and the C-track between positions -14 and -17 were important for specific E sigma32 recognition. Western blots showed that sigma32 was only detectable in the exponential phase, and sigma38 appeared in the early stationary phase. In the rpoH mutant KY1429, sigma38 was already present in the exponential growth phase both free and bound to the RNA polymerase core, in good correlation with the transcription levels found. Pm seems to be optimized for recognition by sigma32 as an initial response to the addition of effector to the medium and allows binding of the adaptable sigma38-dependent RNA polymerase in the stationary phase. XylS is always required for Pm transcription. Therefore, the mechanism that controls Pm expression involves specific nucleotide sequences, the abundance of free and core-bound sigma32 and sigma38 factors during growth, and the presence of the regulator activated by an effector.