RNA Polymerase Holoenzymes Can Share a Single Transcription Start Site for the Pm Promoter

Abstract

The Pm promoter of the benzoate meta-cleavage pathway is transcribed with E 32 or E 38 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 38 and 32 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 38 , whereas positions ؊8, ؊9, and the C-track between positions ؊14 and ؊17 were important for specific E 32 recognition. Western blots showed that 32 was only detectable in the exponential phase, and 38 appeared in the early stationary phase. In the rpoH mutant KY1429, 38 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 32 as an initial response to the addition of effector to the medium and allows binding of the adaptable 38 -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 32 and 38 factors during growth, and the presence of the regulator activated by an effector.

Transcriptional Tradeoff between Metabolic and Stress-response Programs in Pseudomonas putida KT2440 Cells Exposed to Toluene

“…Fig. 6 shows an increase in 32 protein in extracts from cells exposed to the aromatics in comparison to control extracts, thus confirming stabilization of this key factor (52,53).…”

“…We entertain the notion that exposure to o-xylene and toluene (and to a lesser extent, to 3MB) rapidly activates the HS 32 protein (Fig. 6 and Table 5) (52,53), which may then take over much of the roaming RNAP and redirect the response toward the defense against damage to the cell architecture (54,58). In this scenario of limited RNAP availability, other metabolic and stress endurance functions can be expressed if other promoters are liberated.…”

The Logic of Decision Making in Environmental Bacteria

“…[79][80][81] that adjust the outputs of each of steps to the growth or stress conditions of the cells. These signals are entered through numerous host factors and endogenous signal molecules: IHF, HU, TurA, PprA, Crc, ppGpp, sigma factor competition, Entner-Doudoroff metabolites, and perhaps several others [73,75,77,78,[82][83][84][85].…”

“…Note that for this analysis, we consider XylSh and XylSi as separate TFs, equally competent for activating Pm. While this distinction may not be mechanistically accurate [85], it allows us to separate the activation of Pm resulting from the master regulator XylR from that brought about by the formation of 3MB after the action of the upper pathway enzymes on m-xylene (see below). It has been possible to merge both Boolean approaches with dynamic modeling for representing the Pr/Ps node, as the lack of information about the TFs makes it necessary to use (if nothing else) a 0/1 approach to describe the interaction between, for example, Eσ 70 , Eσ 54 , and HU and the target promoters [87,88].…”

“…Transcription initiation in this case can be elicited by any of the active forms of XylS (XylSa or XylSh; [92,96]). Moreover, the core RNA Polymerase (RNAP) that activates Pm can employ any of the three sigma factors of the Eσ 54 family, that is, the housekeeping Eσ 70 (RpoD), the stationary phase Eσ 38 (RpoS), and the heat shock Eσ 32 (RpoH; [82,85,97]). This scenario for Pm regulation can be depicted as a gate with five possible inputs and one single output, the binary computation of which can be broken down to discrete OR and AND gates as shown in Figure 15.4b.…”