The archaeal transcriptional initiation machinery closely resembles core elements of the eukaryal polymerase II system. However, apart from the established basal archaeal transcription system, little is known about the modulation of gene expression in archaea. At present, no obvious eukaryal-like transcriptional regulators have been identified in archaea. Instead, we have previously isolated an archaeal gene, the Pyrococcus furiosus lrpA, that potentially encodes a bacterial-like transcriptional regulator. In the present study, we have for the first time addressed the actual involvement of an archaeal Lrp homologue in transcription modulation. For that purpose, we have produced LrpA in Escherichia coli. In a cell-free P. furiosus transcription system we used wild-type and mutated lrpA promoter fragments to demonstrate that the purified LrpA negatively regulates its own transcription. In addition, gel retardation analyses revealed a single protein-DNA complex, in which LrpA appeared to be present in (at least) a tetrameric conformation. The location of the LrpA binding site was further identified by DNaseI and hydroxyl radical footprinting, indicating that LrpA binds to a 46-base pair sequence that overlaps the transcriptional start site of its own promoter. The molecular basis of the transcription inhibition by LrpA is discussed.Recent studies have revealed that the archaeal transcriptional machinery represents a simplified version of the eukaryal RNA polymerase II transcription apparatus, which involves homologues of the TATA-binding protein (TBP), 1 the transcription factor IIB (TFIIB; the archaeal homologue is called TFB), and the multi-subunit RNA polymerase II (for a recent review, see Ref. 1). The initiation process starts when the TBP interacts specifically with the core promoter element, the TATA box, which is located at positions Ϫ25 to Ϫ30 relative to the transcriptional start site (ϩ1). This complex is stabilized by TFB, which interacts with TBP as well as with the nucleotides Ϫ42 to Ϫ19 that flank the TATA box (2). In particular, a sequence upstream of the TATA box (called the TFB-responsive element or BRE) is essential for transcriptional polarity (3,4

Vanadium haloperoxidases and the bacterial class A nonspecific acid phosphatases have a conserved active site. It is shown that vanadate-substituted recombinant acid phosphatase from Shigella flexneri (PhoN-Sf) and Salmonella enterica ser. typhimurium (PhoN-Se) in the presence of H 2 O 2 are able to oxidize bromide to hypobromous acid. Vanadate is essential for this activity. The kinetic parameters for the artificial bromoperoxidases have been determined. The K m value for H 2 O 2 is about the same as that for the vanadium bromoperoxidases from the seaweed Ascophyllum nodosum. However, the K m value for Br -is about 10-20 times higher, and the turnover values of about 3.4 min )1 and 33 min )1 for PhoN-Sf and PhoN-Se, respectively, are much slower, than those of the native bromoperoxidase. Thus, despite the striking similarity in the active-site structures of the vanadium haloperoxidases and the acid phophatase, the turnover frequency is low, and clearly the active site of acid phosphatases is not optimized for haloperoxidase activity. Like the native vanadium bromoperoxidase, the vanadate-substituted PhoN-Sf and PhoN-Se catalyse the enantioselective sulfoxidation of thioanisole.Keywords: acid phosphatase; brominating activity; enantioselective sulfoxidation; vanadium bromoperoxidase; vanadium chloroperoxidase.Vanadium haloperoxidases are enzymes that catalyse the oxidation of a halide by hydrogen peroxide to the corresponding hypohalous acids according to:The enzymes are named after the most electronegative halide ion they are able to oxidize, therefore chloroperoxidase oxidizes Cl -, Br -, I -and bromoperoxidase oxidizes Brand I -. This class of enzymes binds vanadate (HVO 4 2-) as a prosthetic group [1,2]. It is possible to prepare an apo form of these enzymes which is re-activated by vanadate. This re-activation is competitively inhibited by structural analogues of vanadate (tetrahedral compounds) such as phosphate and molybdate [3,4]. The crystal structures [5][6][7] of vanadium chloroperoxidase and bromoperoxidase from fungus Curvularia inaequalis and the seaweed Ascophyllum nodosum show that vanadate in these enzymes is covalently attached to a histidine residue while five residues donate hydrogen bonds to the nonprotein oxygens. The resulting structure shown for the chloroperoxidase (Fig. 1A) is that of a trigonal bipyramid with three nonprotein oxygens in the equatorial plane which are hydrogen-bonded to Arg360, Arg490, Lys353, Ser402, and Gly403. The fourth oxygen (hydroxide group) at the apical position is hydrogen-bonded to His404. The nitrogen atom from a histidine residue (His496) is at the other apical position. The above vanadatebinding amino acids were shown to be conserved in two bromoperoxidases from seaweed and several acid phosphatases among the large group of soluble bacterial nonspecific class A acid phosphatases [5,[7][8][9][10][11][12]. Examples are the nonspecific acid phosphatase from Shigella flexneri (PhoN-Sf) and the enzyme from Salmonella enterica ser. typhimurium (PhoN-Se) [13,14]....

The ppc gene, encoding phosphoenolpyruvate carboxylase (PEPC), from Rhodopseudomonas palustris No. 7 was cloned and sequenced. Primer extension analysis identified a transcriptional start site 42 bp upstream of the ppc initiation codon. An R. palustris No. 7 PEPC-deficient strain showed a slower doubling time compared with the wild-type strain either anaerobically in the light or aerobically in the dark, when pyruvate was used as a carbon source.

In Escherichia coli, cAMP levels vary with the carbon source used in the culture medium. These levels are dependent on the cellular concentration of phosphorylated EnzymeIIAglc, a component of the glucose-phosphotransferase system, which activates adenylate cyclase (AC). When cells are grown on glucose 6-phosphate (Glc6P), the cAMP level is particularly low. In this study, we investigated the mechanism leading to the low cAMP level when Glc6P is used as the carbon source, i.e. the mechanism preventing the activation of AC by phosphorylated EnzymellAglc. Glc6P is transported via the Uhp system which is inducible by extracellular Glc6P. The Uhp system comprises a permease UhpT and three proteins UhpA, UhpB and UhpC which are necessary for uhpT gene transcription. Controlled expression of UhpT in the absence of the regulatory proteins (UhpA, UhpB and UhpC) allowed us to demonstrate that (i) the Uhp regulatory proteins do not prevent the activation of AC by direct interaction with EnzymellAglc and (ii) an increase in the amount of UhpT synthesized (corresponding to an increase in the amount of Glc6P transported) correlates with a decrease in the cAMP level. We present data indicating that Glc6P per se or its degradation is unlikely to be responsible for the low cAMP level. It is concluded that the level of cAMP in the cell is determined by the flux of Glc6P through UhpT.

No abstract