Recruitment of σ54-RNA Polymerase to the Pu Promoter of Pseudomonas putida through Integration Host Factor-mediated Positioning Switch of α Subunit Carboxyl-terminal Domain on an UP-like Element

Abstract

The interactions between the 54 -containing RNA polymerase ( 54 -RNAP) and the region of the Pseudomonas putida Pu promoter spanning from the enhancer to the binding site for the integration host factor (IHF) were analyzed both by DNase I and hydroxyl radical footprinting. A short Pu region centered at position ؊104 was found to be involved in the interaction with 54 -RNAP, both in the absence and in the presence of IHF protein. Deletion or scrambling of the ؊104 region strongly reduced promoter affinity in vitro and promoter activity in vivo, respectively. The reduction in promoter affinity coincided with the loss of IHF-mediated recruitment of the 54 -RNAP in vitro. The experiments with oriented-␣ 54 -RNAP derivatives containing bound chemical nuclease revealed interchangeable positioning of only one of the two ␣ subunit carboxylterminal domains (␣CTDs) both at the ؊104 region and in the surroundings of position ؊78. The addition of IHF resulted in perfect position symmetry of the two ␣CTDs. These results indicate that, in the absence of IHF, the 54 -RNAP asymmetrically uses only one ␣CTD subunit to establish productive contacts with upstream sequences of the Pu promoter. In the presence of IHFinduced curvature, the closer proximity of the upstream DNA to the body of the 54 -RNAP can allow the other ␣CTD to be engaged in and thus favor closed complex formation.Bacterial promoters are modular DNA regions able to establish productive interactions both with subunits of RNA polymerase holoenzyme (RNAP 1 ; subunit composition: ␣ 2 ␤␤Ј ) and regulatory proteins (1-4). The core promoter elements are signature tags for factor selectivity (4). The major sigma factor 70 generally directs RNAP to interact with the core DNA elements Ϫ10 and Ϫ35 (hexamers with consensus 5Ј-TATAAT-3Ј and 5Ј-TTGACA-3Ј, respectively) (5). The core promoter elements can be both overlapped and flanked by protein-bound DNA sites involved in the fine modulation of promoter activity (2, 4). In the last decade, a considerable amount of attention has been given to a A ϩ T-rich promoter sequence, the UP element, located upstream of the core promoter region and consisting of two distinct subsites, each of which, by itself, can be bound by the carboxyl-terminal domain of the RNAP ␣ subunit (␣CTD) (3, 6 -10). ␣CTD recognizes and interacts with the backbone structure in the minor groove of the UP element. The A ϩ T-rich sequence of the UP element are needed to provide the optimum width of minor groove for interaction with ␣CTD (7, 11). Several lines of evidence showed that the role of the UP elements is to stimulate transcription in an activator protein-independent manner and to a different extent (from 1.5 to ϳ90-fold) depending on the similarity with the consensus UP element sequence (3, 9). It is currently believed that the transcription stimulation by an UP element has to be traced mainly to the cooperation of the sigma factor and the ␣ subunit in RNAP binding to the promoter (1, 12). Thus, the presence of an UP element in a promoter plays the major ...

Role of Integration Host Factor in the Transcriptional Activation of Flagellar Gene Expression in Caulobacter crescentus

“…6A). The activity of both of these increased approximately twofold in the flbD::Tn5 strain relative to wild-type, similar to what has been previously reported for these class II promoters (35). Interestingly, promoter activity of reporter gene fusions of the two genes divergently transcribed from fliL and fliX, the class III flgF and flgI genes (Fig.…”

“…Pure populations of wild-type and ⌬ihfB swarmer cells containing a class III, fliK-lacZ transcriptional reporter fusion were isolated and allowed to progress synchronously through the cell cycle in M2 minimal medium. At the indicated time points a portion of the culture was pulse-labeled with 35 STrans-label for 5 min followed by an ice-cold methionine chase. The labeled reporter fusion was immunoprecipitated using anti-␤-galactosidase antibody from portions of each time point sample containing equal amounts of labeled protein and separated by SDS-PAGE.…”

“…Experiments with the Pseudomonas putida Pu promoter have shown that IHF can also function to recruit RNA polymerase holoenzyme to promoter sequences (7,35). IHF has been demonstrated to be required for the transcription of the 54 nifH promoter (26) and C. crescentus class III and IV flagellar promoters in in vitro experiments (5).…”

Genetic Evidence that Catabolites of the Entner-Doudoroff Pathway Signal C Source Repression of the σ54PuPromoter ofPseudomonas putida

“…First, there are inhibitory effects due to the growth rate and general energy load of the cells. This phenomenon, which is maximally manifest during exponential growth in Luria-Bertani medium (20), results from a combination of growth phase-dependent expression of IHF (which is required for Pu activity (29,37,49), with sigma factor competition in stationary phase mediated by (p)ppGpp (22,26,48). These elements ultimately limit the engagement of the 54 -RNAP with the Ϫ12 to Ϫ24 region of the promoter, which is weak during exponential growth and effective only at the onset of stationary phase (49).…”

σ54-Promoter Discrimination and Regulation by ppGpp and DksA

“…2A, except that maximal transcriptional output was reduced by lack of IHF-binding (19) and Pu (52) are shown in bold and aligned with the consensus sequence that includes the core 5Ј-WATCAR-TTR-3Ј motif (where W is A or T and R is A or G) separated from a less conserved A/T-rich tract of 4 to 6 bp (lowercase letters) (53). The residues shuffled to disrupt the core IHF consensus in the xh-Po(-IHF)/Px promoter series are underlined, as are the bases rendered hydroxyl radical hypersensitive by binding of the ␣-subunits of 54 -RNAP in the Pu UP-element (40). The lower part of the figure shows the DNA sequences of the Ϫ39 to ϩ2 regions of the indicated 54 -promoters, with nucleotides that differ from Po in the Ϫ33 to ϩ2 region underlined.…”