Transcription activation by FNR: evidence for a functional activating region 2

Abstract

The FNR protein of Escherichia coli controls the transcription of target genes in response to anoxia via the assembly-disassembly of oxygen-labile iron-sulfur clusters. Previous work identified patches of surface-exposed amino acids (designated activating regions 1 and 3 [AR1 and AR3, respectively]) of FNR which allow it to communicate with RNA polymerase (RNAP) and thereby activate transcription. Previously it was thought that FNR lacks a functional activating region 2 (AR2), although selecting for mutations that compensate for defective AR1 or a miscoordinated iron-sulfur cluster can reactivate AR2. Here we show that the substitution of two surface-exposed lysine residues (Lys49 and Lys50) of FNR impaired transcription from class II (FNR box centered at -41.5) but not class I (FNR box centered at -71.5) FNR-dependent promoters. The degree of impairment was greater when a negatively charged residue (Glu) replaced either Lys49 or Lys50 than when uncharged amino acid Ala was substituted. Oriented heterodimers were used to show that only the downstream subunit of the FNR dimer was affected by the Lys-->Ala substitutions at a class II promoter. Site-directed mutagenesis of a negatively charged patch ((162)EEDE(165)) within the N-terminal domain of the RNAP alpha subunit that interacts with the positively charged AR2 of the cyclic AMP receptor protein suggested that Lys49 and Lys50 of FNR interact with this region of the alpha subunit of RNAP. Thus, it was suggested that Lys49 and Lys50 form part of a functional AR2 in FNR.

FIG. 1.

Locations of activating regions in FNR. The predicted structure of an FNR monomer based on CRP (25) shows the locations of the previously defined activating regions: AR1, responsible for contacting αCTD, and AR3, responsible for contacting σ⁷⁰. The locations of lysine residues (K49 and K50) that form part of the newly identified AR2 are indicated. Also shown are the N and C termini, α helices (cylinders A to F), β strands (arrows 1 to 12), and essential cysteine residues (C20, C23, C29, and C122) that act as ligands for the oxygen-labile [4Fe-4S] cluster.

FIG. 2.

Organization of simple class I and class II FNR-dependent promoters. (A) At class I promoters, FNR binds to a site centered at −61.5 or further upstream. AR1 of the downstream subunit of the FNR dimer contacts αCTD (▪). (B) At class II promoters, FNR binds to a site centered at or near −41.5 and is thus embedded within RNAP; multiple interactions are possible. Two FNR-RNAP contacts were previously characterized at class II promoters. The AR1 surface of the upstream subunit of the FNR dimer contacts αCTD (▪), and the AR3 surface of the downstream subunit of FNR contacts σ⁷⁰ (⧫). Also indicated is the contact between AR2 and αNTD (▴), which is used by CRP and now shown to be present in FNR.

FIG. 3.

Partial rescue of impaired AR2 by amino acid substitutions in αNTD (RpoA). All strains carried a low-copy-number plasmid with the simple FNR-activated class II promoter, FF(−41.5), fused to lacZ (32) and plasmid pBR322-encoded wild-type FNR or FNR-K49E. In addition, RpoA or the indicated variants were supplied on plasmids (Table 1) encoding wild-type E165, A165, or K165. All cultures were grown under anaerobic conditions in L broth supplemented with glucose (0.2%) at 37°C for 16 h. β-Galactosidase activity was measured in at least three independent cultures of each strain.