Additional Determinants within Escherichia coli FNR Activating Region 1 and RNA Polymerase α Subunit Required for Transcription Activation

Abstract

The global anaerobic regulator FNR is a DNA binding protein that activates transcription of genes required for anaerobic metabolism in Escherichia coli through interactions with RNA polymerase (RNAP). Alaninescanning mutagenesis of FNR amino acid residues 181 to 193 of FNR was utilized to determine which amino acid side chains are required for transcription of both class II and class I promoters. In vivo assays of FNR function demonstrated that a core of residues (F181, R184, S187, and R189) was required for efficient activation of class II promoters, while at a class I promoter, FF(؊61.5), only S187 and R189 were critical for FNR activation. Site-directed mutagenesis of positions 184, 187, and 189 revealed that the positive charge contributes to the function of the side chain at positions 184 and 189 while the serine hydroxyl is critical for the function of position 187. Subsequent analysis of the carboxy-terminal domain of the ␣ subunit (␣CTD) of RNAP, using an alanine library in single copy, revealed that in addition to previously characterized side chains (D305, R317, and L318), E286 and E288 contributed to FNR activation of both class II and class I promoters, suggesting that ␣CTD region 285 to 288 also participates in activation by FNR. In conclusion, this study demonstrates that multiple side chains within region 181 to 192 are required for FNR activation and the surface of ␣CTD required for FNR activation is more extensive than previously observed.A common mechanism of activation by transcription factors employs a series of macromolecular interactions, from binding DNA to multiple contacts with RNA polymerase (RNAP), which allows target promoters to overcome intrinsic defects in transcription initiation (21). This study focused on the Escherichia coli global anaerobic regulator FNR and the protein requirements essential for transcription activation. In particular, this study further investigated the residues of FNR that have been proposed to interact with the ␣ subunit of RNAP.FNR, which regulates transcription in response to O 2 deprivation, belongs to a family of transcriptional regulators related to the cyclic AMP receptor protein, CRP (13). In its active form, FNR is a homodimer (16) which protects an ϳ22-bp sequence in DNase I footprinting experiments (9, 14). The vast majority of FNR-activated promoters contain an FNR binding site centered approximately 41.5 bp upstream of the transcriptional start site and are termed class II promoters (Fig. 1A) (6). At this position, FNR is able to make multiple contacts with RNAP through three domains: activating region 1 (AR1), AR2, and AR3 (3,5,14,15,31,33,34). FNR-AR3 is active in the downstream subunit (3) and is likely to contact the 70 subunit of RNAP (23). FNR-AR2, which plays a minor role in activation, is also active in the downstream subunit (5), but its interaction partner is proposed to be the amino-terminal domain of the ␣ subunit of RNAP (5). FNR-AR1 is active in the upstream subunit (3, 33) and is proposed to interact with the carboxy-terminal dom...

ClpXP-dependent Proteolysis of FNR upon Loss of its O2-sensing [4Fe–4S] Cluster

“…9 In the dimerized, [4Fe-4S] 2C -containing state, FNR binds site-specifically to DNA and interacts with RNA polymerase (RNAP) to regulate transcription from FNR-dependent promoters. 7,[9][10][11][12][13][14][15][16][17][18][19][20][21][22] Both in vivo and in vitro studies have demonstrated that under aerobic conditions, the [4Fe-4S] 2C cluster is converted to a [2Fe-2S] 2C cluster, resulting in an inactive, monomeric form of FNR ([2Fe-2S] 2C -FNR). 8,10,23,24 The [2Fe-2S] 2C cluster is further destroyed under aerobic growth conditions, presumably as a result of its instability to superoxide, a by-product of aerobic metabolism, as demonstrated in vitro.…”

Anaerobic Catabolism of Aromatic Compounds: a Genetic and Genomic View

“…Whereas these four cysteine residues are perfectly conserved in AcpR from Azoarcus (at positions 18, 21, 27, and 122), in the AadR protein, only two cysteine residues (at positions 20 and 117) are conserved. The AR-1, AR-2, and AR-3 activation regions, which are involved in the interaction of the E. coli FNR protein with the ␣-C-terminal domain, ␣-N-terminal domain, and 70 -C-terminal domain subunits of RNAP, respectively (242,371), are also conserved in AcpR and AadR, although in the latter, there is low amino acid sequence conservation at the AR-1 region (97). A new branch of the FNR/CRP superfamily that has recently emerged includes transcriptional regulators such as CprK ( Table 3) that mediate the response to halogenated aromatic compounds in the dehalorespiration of different Desulfitobacterium strains (286).…”

Salmonella spp. survival strategies within the host gastrointestinal tract

“…Fnr, also named OxrA in Salmonella, is a cytoplasmic oxygen sensor that can bind promoter sequences, and interacts with the RpoA subunit of RNA polymerase to increase the efficiency of transcription of a variety of genes required for anerobic metabolism, while it represses many of the genes encoding enzymes involved in aerobic electron transport, oxidative phosphorylation and some tricarboxylic cycle enzymes (Unden et al, 1995;Wei & Miller, 1999;Perrenoud & Sauer, 2005;Weber et al, 2005).…”