Role of Activating Region 1 of Escherichia coliFNR Protein in Transcription Activation at Class II Promoters

Wing, Helen J.; Green, Jeff; Guest, John R.; Busby, Stephen J. W.

doi:10.1074/jbc.m000390200

Cited by 37 publications

(52 citation statements)

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“…The AR1 contact of CRP enhances the binding of RNAP to class II promoters, and the AR2 contact directly activates transcription by increasing the rate of open complex formation (19,22). The AR1 contact of FNR accelerates the transition from the closed to the open complex (31), and AR3 is also thought to provide direct activation by interaction with 70 (15). The evidence indicating that AR2 of FNR interacts with the same region of ␣NTD as CRP suggests that AR2 probably fulfills the same function in both regulators.…”

Section: Discussionmentioning

confidence: 99%

“…The AR1 contact is required for transcription activation at class I promoters (where the FNR box is located close to position Ϫ61 or further upstream), where contact is established between AR1 of the downstream subunit of the FNR dimer and ␣CTD (32). The AR1 contact is also used at class II promoters (where the FNR box is located at Ϫ41); however, in this situation it is the upstream subunit of the FNR dimer that makes contact with ␣CTD to promote transcription (8,31). In addition to the AR1 contact, FNR has a second interaction with RNAP at class II promoters (32).…”

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Transcription Activation by FNR: Evidence for a Functional Activating Region 2

et al. 2002

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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 Lys3Ala 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 ␣ 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 ␣ subunit of RNAP. Thus, it was suggested that Lys49 and Lys50 form part of a functional AR2 in FNR.The Escherichia coli FNR protein is a global transcription factor that controls the expression of target genes in response to oxygen starvation. It is structurally related to the cyclic AMP receptor protein (CRP), except for the presence of an Nterminal extension that contains three of the four cysteine residues that are essential for normal FNR function: C20, C23, C29, and C122 (17, 26). The essential cysteine residues are presumed to act as ligands for an oxygen-labile [4Fe-4S] cluster (6,7,10). Anaerobic acquisition of the iron-sulfur cluster converts monomeric FNR into a dimeric form containing two [4Fe-4S] clusters (10). This transition to the dimeric state enhances site-specific DNA binding and mediates transcription regulation by establishing direct FNR-RNA polymerase (RNAP) contacts (1,2,4,5,9,13,15,29).The activating contacts between FNR and RNAP involve two surface-exposed regions of FNR designated activating region 1 (AR1), which contacts the C-terminal domain of the ␣ subunit of RNAP (␣CTD) (13, 29), and activating region 3 (AR3), which contacts 70 (9, 15) (Fig. 1). The contacts established between FNR and RNAP depend upon the architecture of particular promoters (Fig. 2). The AR1 contact is required for transcription activation at class I promoters (where the FNR box is located close to position Ϫ61 or further upstream), where contact is established between AR1 of the downstream subunit of the FNR dimer and ␣CTD (32). The AR1 contact is also used at class II promoters (where the FNR box is loca...

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Section: Discussionmentioning

confidence: 99%

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Transcription Activation by FNR: Evidence for a Functional Activating Region 2

et al. 2002

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“…In addition to the FNRdependent increase in the affinity of RNAP for the P N promoter, in vitro transcription experiments revealed that FNR is essential for the initiation of transcription at some step after RNAP-P N closed-complex formation. As has been shown for other FNR-dependent promoters (376), FNR may activate the transcription of genes controlled by P N by promoting isomerization from the transcriptionally inactive closed complex to the transcriptionally active open complex (97).…”

Section: Overimposed Regulationmentioning

confidence: 99%

Anaerobic Catabolism of Aromatic Compounds: a Genetic and Genomic View

Carmona

Zamarro

Blázquez

et al. 2009

Microbiol Mol Biol Rev

392

381

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SUMMARY Aromatic compounds belong to one of the most widely distributed classes of organic compounds in nature, and a significant number of xenobiotics belong to this family of compounds. Since many habitats containing large amounts of aromatic compounds are often anoxic, the anaerobic catabolism of aromatic compounds by microorganisms becomes crucial in biogeochemical cycles and in the sustainable development of the biosphere. The mineralization of aromatic compounds by facultative or obligate anaerobic bacteria can be coupled to anaerobic respiration with a variety of electron acceptors as well as to fermentation and anoxygenic photosynthesis. Since the redox potential of the electron-accepting system dictates the degradative strategy, there is wide biochemical diversity among anaerobic aromatic degraders. However, the genetic determinants of all these processes and the mechanisms involved in their regulation are much less studied. This review focuses on the recent findings that standard molecular biology approaches together with new high-throughput technologies (e.g., genome sequencing, transcriptomics, proteomics, and metagenomics) have provided regarding the genetics, regulation, ecophysiology, and evolution of anaerobic aromatic degradation pathways. These studies revealed that the anaerobic catabolism of aromatic compounds is more diverse and widespread than previously thought, and the complex metabolic and stress programs associated with the use of aromatic compounds under anaerobic conditions are starting to be unraveled. Anaerobic biotransformation processes based on unprecedented enzymes and pathways with novel metabolic capabilities, as well as the design of novel regulatory circuits and catabolic networks of great biotechnological potential in synthetic biology, are now feasible to approach.

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“…The [4Fe-4S] form of FNR acts as both a positive and a negative regulator of gene expression, activating transcription by recruiting RNA polymerase, or repressing transcription by inhibiting the formation of productive promoter-RNA polymerase interactions (Barnard et al, 2004;Bell & Busby, 1994;Blake et al, 2003;Browning et al, 2003;Green & Marshall, 1999;Green et al, 1998;Lamberg & Kiley, 2000;Lamberg et al, 2002;Li et al, 1998; Lonetto et al, 1998;Marshall et al, 2001;Meng et al, 1997;Williams et al, 1997;Wing et al, 2000). Exposure of E. coli to air is sensed by the , 2005).…”

Section: Introductionmentioning

confidence: 99%

“…Accordingly, deletion of the iscS gene (a cysteine desulphurase that provides the sulphur for iron-sulphur cluster assembly) impairs FNR activity in vivo (Schwartz et al, 2000). Furthermore, comparison of the effects on FNR, which requires an iron-sulphur cluster for activity, and those on FNR*, an FNR variant that is active in the absence of an iron-sulphur cluster, suggests that the isc operon plays a major role in the assembly of the FNR ironsulphur cluster (Schwartz et al, 2000).The [4Fe-4S] form of FNR acts as both a positive and a negative regulator of gene expression, activating transcription by recruiting RNA polymerase, or repressing transcription by inhibiting the formation of productive promoter-RNA polymerase interactions (Barnard et al, 2004;Bell & Busby, 1994;Blake et al, 2003;Browning et al, 2003;Green & Marshall, 1999;Green et al, 1998;Lamberg & Kiley, 2000;Lamberg et al, 2002;Li et al, 1998; Lonetto et al, 1998;Marshall et al, 2001;Meng et al, 1997;Williams et al, 1997;Wing et al, 2000). Exposure of E. coli to air is sensed by the , 2005).…”

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In vivo cycling of the Escherichia coli transcription factor FNR between active and inactive states

Dibden

Green

2005

Microbiology

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FNR proteins are transcription regulators that sense changes in oxygen availability via assembly-disassembly of [4Fe-4S] clusters. The Escherichia coli FNR protein is present in bacteria grown under aerobic and anaerobic conditions. Under aerobic conditions, FNR is isolated as an inactive monomeric apoprotein, whereas under anaerobic conditions, FNR is present as an active dimeric holoprotein containing one [4Fe-4S] cluster per subunit. It has been suggested that the active and inactive forms of FNR are interconverted in vivo, or that iron-sulphur clusters are mostly incorporated into newly synthesized FNR. Here, experiments using a thermo-inducible fnr expression plasmid showed that a model FNR-dependent promoter is activated under anaerobic conditions by FNR that was synthesized under aerobic conditions. Immunoblots suggested that FNR was more prone to degradation under aerobic compared with anaerobic conditions, and that the ClpXP protease contributes to this degradation. Nevertheless, FNR was sufficiently long lived (half-life under aerobic conditions,~45 min) to allow cycling between active and inactive forms. Measuring the abundance of the FNR-activated dms transcript when chloramphenicol-treated cultures were switched between aerobic and anaerobic conditions showed that it increased when cultures were switched to anaerobic conditions, and decreased when aerobic conditions were restored. In contrast, measurement of the abundance of the FNR-repressed ndh transcript under the same conditions showed that it decreased upon switching to anaerobic conditions, and then increased when aerobic conditions were restored. The abundance of the FNR-and oxygen-independent tatE transcript was unaffected by changes in oxygen availability. Thus, the simplest explanation for the observations reported here is that the FNR protein can be switched between inactive and active forms in vivo in the absence of de novo protein synthesis. INTRODUCTIONThe FNR protein is an oxygen-responsive transcription regulator that controls the expression of more than 100 transcriptional units in Escherichia coli, and coordinates the transition from aerobic to anaerobic growth (Becker et al., 1996;Covert et al., 2004; Gonzalez et al., 2003;Guest et al., 1996;Kang et al., 2005; Salmon et al., 2003;Sawers, 1999;Unden & Bongaerts, 1997). The intracellular concentration of FNR is similar under both aerobic and anaerobic conditions (Sutton et al., 2004a;Unden & Duchene, 1987), but FNR is activated under anaerobic conditions by the acquisition of [4Fe-4S] clusters that promote the formation of homodimers, and enhance DNA binding at sites resembling the consensus sequence TTGAT-N 4 -ATCAA (Bauer et al., 1999;Eiglmeier et al., 1989;Green et al., 1996Green et al., , 2001Green & Paget, 2004;Khoroshilova et al., 1995Khoroshilova et al., , 1997Kiley & Beinert, 1999Lazazzera et al., 1993 Lazazzera et al., , 1996Moore & Kiley, 2001;Patschkowski et al., 2000;Popescu et al., 1998). Four essential cysteine residues (Cys20, 23, 29 and 122) ligate the FNR [4Fe-4S] ...

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Role of Activating Region 1 of Escherichia coliFNR Protein in Transcription Activation at Class II Promoters

Cited by 37 publications

References 27 publications

Transcription Activation by FNR: Evidence for a Functional Activating Region 2

Transcription Activation by FNR: Evidence for a Functional Activating Region 2

Anaerobic Catabolism of Aromatic Compounds: a Genetic and Genomic View

In vivo cycling of the Escherichia coli transcription factor FNR between active and inactive states

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