The structure of a transcription activation subcomplex reveals how σ⁷⁰is recruited to PhoB promoters

Abstract

PhoB is a two-component response regulator that activates transcription by interacting with the r 70 subunit of the E. coli RNA polymerase in promoters in which the À35 r 70 -recognition element is replaced by the pho box. The crystal structure of a transcription initiation subcomplex that includes the r 4 domain of r 70 fused with the RNA polymerase b subunit flap tip helix, the PhoB effector domain and the pho box DNA reveals how r 4 recognizes the upstream pho box repeat. As with the À35 element, r 4 achieves this recognition through the N-terminal portion of its DNA recognition helix, but contact with the DNA major groove is less extensive. Unexpectedly, the same recognition helix contacts the transactivation loop and helices a2 and a3 of PhoB. This result shows a simple and elegant mechanism for polymerase recruitment to pho box promoters in which the lost À35 element contacts are compensated by new ones with the activator. In addition, r 4 is reoriented, thereby suggesting a remodelling mechanism for transcription initiation.

PhoB transcriptional activator binds hierarchically to pho box promoters

“…PhoB E has a winged-helix motif for DNA binding, where helix α 3 is the recognition helix. Between helices α 2 and α 3 within the modified helix-turn-helix motif, there is a seven-residue loop which has been named the transactivation loop because it was postulated to interact with the σ 70 subunit to activate transcription (Makino et al , 1996 ;Blanco et al , 2002 ); we recently confirmed this theory with the crystallographic structure of a ternary complex formed by the PhoB E dimer, a pho box DNA and the σ 4 domain of σ 70 (Blanco et al , 2011 ). In the present structure, a tandem of two protomers arranged head-to-tail sits at one side of the DNA molecule, the recognition helices α 3 lay along the major groove while the wing tips contact the downstream minor groove, performing specific (with bases) and nonspecific (with phosphates) interactions ( Figure 3B).…”

“…σ 70 is formed by three structural domains ( σ 2 , σ 3 and σ 4 ), the C-terminal region ( σ 4 ) being the one that recognizes the -35 element and hence the putative PhoB E target for activating transcription (Makino et al , 1993 ;Kumar et al , 1994 ). In a recent study we provided crystallographic evidence that the DNA-bound PhoB E domain directly contacts the σ 4 domain of σ 70 to accomplish RNA polymerase recruitment (Blanco et al , 2011 ). Here we complement this finding with biochemical data on the PhoB mode of binding to natural promoters containing multiple pho boxes and report a new σ -free crystal structure of the PhoB E dimer bound in tandem to a 26-mer DNA comprising the pho box promoter sequence.…”

“…PhoB binding to the pho box promoter sequence leads to the recruitment of RNA polymerase. The DNA-PhoB complex improves the interaction with the σ 4 domain of σ 70 (Blanco et al , 2011 ) and hence PhoB mediation effectively triggers the expression of the specific genes belonging to the Pho regulon and phosphate uptake is fulfilled.…”

Structure and dynamics of the polymyxin-resistance-associated response regulator PmrA in complex with the promoter DNA

“…To better understand the roles of the REC-DBD interface in transcription activation, we dock the E. coli RNAPH 18 to the PmrA-DNA complex structure on the basis of the crystal structure of the s 4 -b-flap tip helix chimer/PhoB-DBD/DNA ternary complex 29 (Supplementary Fig. 14).…”

The Bordetella pertussis model of exquisite gene control by the global transcription factor BvgA

“…3d) (Benoff et al, 2002) or PhoB at pho box DNA (Fig. 3f) (Blanco et al, 2011), the activator is poised to interact with a common set of region 4 residues (discussed by Bonocora et al, 2008). However, the particular positioning of BvgA~P at P fim3 means that these residues are not available for a region 4/activator interaction.…”