Both fis-dependent and factor-independent upstream activation of the rrnB P1 promoter are face of the helix dependent

Abstract

Transcription from the Escherichia coli rrnB P1 promoter is increased by a cis-acting sequence which extends upstream of the -35 hexamer to about -150 with respect to the transcription initiation site, the Upstream Activation Region (UAR). Activation by the UAR involves two components: (1) a trans-acting protein, Fis, which binds to three sites in the UAR between -60 and -150, and (2) the UAR sequences themselves which affect RNA polymerase (RNAP) activity independent of other proteins. We refer to the latter as Factor-Independent Activation (FIA). In addition to its interactions with the -10 and -35 hexamers typical of E. coli promoters, RNAP makes contacts to the -53 region of rrnB P1, which may be related to the FIA effect. We constructed a series of insertion mutants containing integral and non-integral numbers of helical turns at position -46, between the Fis binding sites and the -35 region, and the resulting promoter activities were measured in vitro and in vivo. The data suggest that both Fis-dependent and factor-independent activation are face of the helix dependent: the Fis binding site and the sequences responsible for factor-independent activation must be correctly oriented relative to RNA polymerase in order to activate transcription. These results, in conjunction with other evidence, support a model for the involvement of direct Fis-RNAP interactions in upstream activation. We also demonstrate that RNAP interacts with the -53 region of the rrnB P1 UAR even when these sequences are displaced upstream of the RNAP binding site, and that these interactions correlate with factor-independent activation.

Real-time footprinting of DNA in the first kinetically significant intermediate in open complex formation by Escherichia coli RNA polymerase

“…Nevertheless, many studies have demonstrated that altering the degree and/or phasing of upstream bending by introducing intrinsically bent DNA sequences (e.g., ref. 31) or by moving binding sites of activators (32) significantly increases (activates) or decreases (represses) the amount of transcription. Fig.…”

“…At 17°C, where the equilibrium constant K 1 is a maximum and conversion of I 1 to I 2 is relatively slow, we previously found that the fraction of promoter DNA in I 1 complexes ( I1 ) at 15 sec after mixing P R promoter with 70 nM (excess) RNAP was 0.55 (6). To increase the fraction of I 1 in the time interval (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) (38) where the only complex predicted to be populated is I 1 (9). Because the signal:noise ratio in these 0°C experiments was marginal, and to avoid the possibility of populating off-pathway complexes at 0°C (39), we identified solution conditions where I 1 is Ͼ70% of promoter DNA (see above) at early times in the time course of RP o formation, and footprinted as follows.…”

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

“…The artificial upstream re-location of the rrnB P1 UP element by a single turn of DNA helix decreases but does not prevent transcription stimulation, while further displacements abolish UP elementdependent transcription (15). The ability of ␣CTD to contact DNA and/or activator molecules at different locations upstream of the core promoter (8,(15)(16)(17)(18)(19)(20)(21) has been attributed to the flexibility of the linker connecting ␣CTD to the ␣ amino-terminal domain (␣NTD) (8,22) assembled in the body of RNAP. This linker flexibility also accounts for the ability of the two copies of ␣CTD to function interchangeably with respect to the subsite recognition within the UP element (10).…”

Localization of the intrinsically bent DNA region upstream of theE.coli rrnBP1 promoter

“…FIS Site I, the site closest to the promoter, accounts for 70 -80% of the effect of FIS on transcription (7). FIS most likely stimulates transcription by interacting directly with RNA polymerase (6,8, and A.J.Bokal, W.Ross, and R.L.Gourse, unpublished results).…”

“…Therefore, if the A/T tracts are responsible for the small degree of curvature associated with this region, either the mutations do not affect this curvature, or the curvature plays little role in the 30-fold increase in transcription resulting from UP element function. On the other hand, either displacement of the UP element by non-integral portions of a helical turn (6,29,34) or creation of an additional T tract with a T substitution at position -37 (29,34) severely decreases promoter activity. Thus, correct positioning of the RNAP alpha subunit (which binds to the UP element) relative to the sigma subunit (which binds to the -10 and -35 hexamers) most likely plays a major role in promoter activity (2).…”