Transcriptional regulation of tyrosine phenol-lyase gene mediated through TyrR and cAMP receptor protein

Using a lac reporter system in Escherichia coli, we showed that the expression of E. herbicola tpl was regulated through TyrR and cAMP receptor protein. Three TyrR boxes upstream of tpl were essential for full expression. The results suggested that the tyrosine-mediated TyrR hexamerization was an important process. The DNA bending between two TyrR boxes, which is triggered by the binding of cAMP receptor protein, may facilitate the conformational change of TyrRs.     

Construction of an Effective Protein Expression System Using the <Emphasis Type="Italic">tpl</Emphasis> Promoter in <Emphasis Type="Italic">Escherichia coli</Emphasis>

An effective protein expression system was constructed in Escherichia coli using the promoter of the tyrosine phenol-lyase (tpl) gene of Erwinia herbicola. This system involves a mutant form of the TyrR protein with an enhanced ability to activate tpl and the TutB protein with an ability to transport L-tyrosine (an inducer of Tpl). The highest expression level obtained for this system was more than twice that obtained for the tac system, although it was lower than the level obtained for the T7 system, as revealed with the lac-reporter assay and SDS-polyacrylamide gel electrophoresis.     

Cyclic AMP Receptor Protein and TyrR Are Required for Acid pH and Anaerobic Induction of hyaB and aniC in Salmonella typhimurium

Two acid-inducible genes, aniC and aciK, that require anaerobiosis and tyrosine for expression were identified as orf326a encoding a potential amino acid/polyamine antiporter and hyaB encoding hydrogenase I, respectively. Cyclic AMP (cAMP) receptor protein, cAMP, and TyrR, regulator of aromatic amino acid metabolism, were strong positive regulators of both genes.     

Isolation of a New Root Growth-stimulating Substance from a Fungus

In the last few decades, enzymatic production of 3,4-dihydroxyphenyl-L-alanine (L-dopa) using tyrosine phenol-lyase (Tpl) has been industrialized. This method has an intrinsic problem of tyrosine contamination because Tpl is synthesized under tyrosine-induced conditions. Herein, we constructed a hyper-L-dopa-producing strain by exploiting a mutant TyrR, an activator of tpl. The highest productivity was obtained for the strain grown under non-induced conditions. It was 30-fold higher than that obtained for tyrosine-induced wild-type cells.     

Specific contacts between residues in the DNA-binding domain of the TyrR protein and bases in the operator of the tyrP gene of Escherichia coli

In the presence of tyrosine, the TyrR protein of Escherichia coli represses the expression of the tyrP gene by binding to the double TyrR boxes which overlap the promoter. Previously, we have carried out methylation, uracil, and ethylation interference experiments and have identified both guanine and thymine bases and phosphates within the TyrR box sequences that are contacted by the TyrR protein (J. S. Hwang, J. Yang, and A. J. Pittard, J. Bacteriol. 179:1051-1058, 1997). In this study, we have used missing contact probing to test the involvement of all of the bases within the tyrP operator in the binding of TyrR. Our results indicate that nearly all the bases within the palindromic arms of the strong and weak boxes are important for the binding of the TyrR protein. Two alanine-substituted mutant TyrR proteins, HA494 and TA495, were purified, and their binding affinities for the tyrP operator were measured by a gel shift assay. HA494 was shown to be completely defective in binding to the tyrP operator in vitro, while, in comparison with wild-Type TyrR, TA495 had only a small reduction in DNA binding. Missing contact probing was performed by using the purified TA495 protein, and the results suggest that T495 makes specific contacts with adenine and thymine bases at the +/-5 positions in the TyrR boxes.     

Mutational analysis of repression and activation of the tyrP gene in Escherichia coli.

In a previous report it had been suggested that the tyrP gene of Escherichia coli may be expressed from two separate promoters. We have endeavored to confirm this suggestion by primer extension studies and the separate subcloning of each of these promoters. In these studies, we found a single promoter whose expression was repressed by TyrR protein in the presence of tyrosine and activated by TyrR protein in the presence of phenylalanine. Two adjacent TYR R boxes, with the downstream one overlapping the tyrP promoter, are the likely targets for the action of TyrR protein. Mutational analysis showed that both TYR R boxes were required for tyrosine-mediated repression but that only the upstream box was required for phenylalanine-mediated activation. In vitro DNase protection studies established that whereas in the absence of tyrosine TyrR protein protected the region of DNA represented by the upstream box, at low TyrR protein concentrations both tyrosine and ATP were required to protect the region of DNA involving the downstream box and overlapping the RNA polymerase binding site.     

Molecular analysis of the TyrR protein-mediated activation of mtr gene expression in Escherichia coli K-12.

Expression of the mtr gene, which encodes a tryptophan-specific transport system in Escherichia coli K-12, is activated by the TyrR protein. Two TyrR protein binding sites (TYR R boxes) are positioned upstream of the -35 promoter region. Mutational and DNase protection studies indicate that TyrR protein binds preferentially to the TYR R box closest to the promoter, and this is essential for activation of gene expression. In the presence of tyrosine and ATP, a second TyrR molecule is able to cooperatively bind to the second box and cause a further increase in the level of activation.     

Importance of the position of TYR R boxes for repression and activation of the tyrP and aroF genes in Escherichia coli.

Tyrosine-mediated repression of aroF and tyrP was studied by inserting DNA sequences between the two adjacent TYR R boxes which, in each case, overlap the respective RNA polymerase binding sites of these genes. In both cases, repression was greatest when homologous regions of these two TYR R boxes were on the same face of the DNA helix and the boxes were directly adjacent. An insertion of 3 bases was sufficient to abolish repression, which was reestablished as the boxes became separated by one full turn of the helix. These observations, coupled with the results of in vitro DNase I protection studies, supported the hypothesis that the binding of TyrR protein to the downstream boxes required cooperative interaction with TyrR protein already bound to the upstream boxes. In the case of tyrP, moving the upstream box also affected activation. Maximal activation was observed when the box was moved 3 or 12 to 14 residues upstream. Practically no activation was seen at intermediate positions, such as +7 and -4. It is hypothesized that these results indicate positions allowing maximal interaction between TyrR protein bound to the upstream box and RNA polymerase bound to the RNA polymerase binding site.     

Critical base pairs and amino acid residues for protein-DNA interaction between the TyrR protein and tyrP operator of Escherichia coli.

In Escherichia coli K-12, the repression of tyrP requires the binding of the TyrR protein to the operator in the presence of coeffectors, tyrosine and ATP. This operator contains two 22-bp palindromic sequences which are termed TyrR boxes. Methylation, uracil, and ethylation interference experiments were used to identify the important sites in the TyrR boxes that make contacts with the TyrR protein. Methylation interference studies demonstrated that guanines at positions +8, -5, and -8 of the strong TyrR box and positions +8, -4, and -8 of the weak box are close to the TyrR protein. Uracil interference revealed that strong van der Waals contacts are made by the thymines at position -7 and +5 of the top strands of both strong and weak boxes and that weaker contacts are made by the thymines at positions +7 (strong box) and -5 and +7 (weak box) of the bottom strand. In addition, ethylation interference suggested that the phosphate backbone contacts are located at the end and central regions of the palindrome. These findings are supported by our results derived from studies of symmetrical mutations of the tyrP strong box. Overall, the results confirm the critical importance of the invariant (G x C)(C x G)8 base pairs for TyrR recognition and also indicate that interactions with (T x A)(A x T)7 are of major importance. In contrast, mutations in other positions result in weaker effects on the binding affinity of TyrR protein, indicating that these positions play a lesser role in TyrR protein recognition. Alanine scanning of both helices of the putative helix-turn-helix DNA-binding motif of TyrR protein has identified those amino acids whose side chains play an essential role in protein structure and DNA binding.     

Regulation of aroL expression by TyrR protein and Trp repressor in Escherichia coli K-12.

The promoter-operator region of the aroL gene of Escherichia coli K-12 contains three TYR R boxes and one TrpR binding site. Mutational analysis showed that TYR R boxes 1 and 3 are essential for TyrR-mediated regulation of aroL expression, while a fully functional TYR R box 2 does not appear to be essential for regulation. Regulation mediated by the TrpR protein required the TYR R boxes and TrpR site to be functional and was observed in vivo only with a tyrR+ strain. Under conditions favoring the formation of TyrR hexamers, DNase I protection experiments revealed the presence of phased hypersensitive sites, indicative of DNA backbone strain. This suggests that TyrR-mediated repression involves DNA looping. Purified TrpR protein protected the putative TrpR binding site in the presence of tryptophan, and this protection was slightly enhanced in the presence of TyrR protein. This result along with the in vivo findings implies that TyrR and TrpR are able to interact in some way. Inserting 4 bp between TYR R box 1 and the TrpR binding site results in increased tyrosine repression and the abolition of the tryptophan effect. Identification of a potential integration host factor binding site and repression studies of a himA mutant support the notion that integration host factor binding normally exerts a negative effect on tyrosine-mediated repression.     

Cyclic AMP-induced p53 destabilization is independent of EPAC in pre-B acute lymphoblastic leukemia cells in vitro

Context: Activation of the tumor suppressor protein p53 facilitates the cellular response to genotoxic stress. Thus, releasing the wild-type p53 from indirect suppression would be crucial to successful killing of cancer cells by DNA-damaging therapeutic agents.

Objective: The aim of this study was to investigate the inhibitory role of cyclic adenosine monophosphate (cAMP) levels on p53 protein in acute lymphoblastic leukemia (ALL) cells. More importantly, we were interested to show through which receptor cAMP acts to promote p53 degradation.

Materials and methods: In cell cultures, we investigated the effects of forskolin/3-isobutyl-1-methylxanthine (IBMX) on stimulated p53 of ALL cell lines. Western blotting analysis was performed to detect the expression of p53, phospho-p53, acetylated-p53, phospho-cAMP response element-binding protein (CREB), and Mdm2 proteins. Flow cytometry was applied to analyze apoptosis. The gene expression of p53 and its target genes was examined by real-time polymerase chain reaction.

Results: We show that elevation of cAMP levels in ALL cells exposed to DNA damage attenuates p53 accumulation. Inhibition of proteosome function with MG-132 reversed the inhibitory effect of cAMP on p53. However, targeting the p53–Mdm2 interaction did not rescue accumulated p53 from the destabilizing signal of cAMP. The specific agonist of the cAMP receptor exchange protein activated by cAMP had no effect on p53 expression in doxorubicin-treated NALM-6 cells, whereas PKA activators decreased p53 accumulation.

Discussion and conclusion: Our studies demonstrate that cAMP-PKA pathway regulates the sensitivity toward DNA-damaging agents via inhibition of a p53-dependent pathway in B-cell precursor ALL (BCP-ALL) cells.     

Cyclic AMP-receptor protein activates aerobactin receptor IutA expression in <Emphasis Type="Italic">Vibrio vulnificus</Emphasis>

The ferrophilic bacterium Vibrio vulnificus can utilize the siderophore aerobactin of Escherichia coli for iron acquisition via its specific receptor IutA. This siderophore piracy by V. vulnificus may contribute to its survival and proliferation, especially in mixed bacterial environments. In this study, we examined the effects of glucose, cyclic AMP (cAMP), and cAMP-receptor protein (Crp) on iutA expression in V. vulnificus. Glucose dose-dependently repressed iutA expression. A mutation in cya encoding adenylate cyclase required for cAMP synthesis severely repressed iutA expression, and this change was recovered by in trans complementing cya or the addition of exogenous cAMP. Furthermore, a mutation in crp encoding Crp severely repressed iutA expression, and this change was recovered by complementing crp. Accordingly, glucose deprivation under iron-limited conditions is an environmental signal for iutA expression, and Crp functions as an activator that regulates iutA expression in response to glucose availability.     

A lowered concentration of cAMP receptor protein caused by glucose is an important determinant for catabolite repression in Escherichia coli

A decreased intracellular concentration of cAMP is insufficient to account for catabolite repression in Escherichia coli. We show that glucose lowers the amount of cAMP receptor protein (CRP) in cells. A correlation exists between CRP and β-galactosidase levels in cells growing under various conditions. Exogenous cAMP completely eliminates catabolite repression in CRP-overproducing cells, while it does not fully reverse the effect of glucose on β-galactosidase expression in wild-type cells. When the CRP concentration is reduced by manipulating the crp gene, β-galactosidase expression decreases in proportion to the concentration of CRP. These findings indicate that the lowered concentration of CRP caused by glucose is one of the major factors for catabolite repression. We propose that glucose causes catabolite repression by lowering the intracellular levels of both CRP and cAMP.     

Pharmacological characterization of a β‐adrenergic‐like octopamine receptor in Plutella xylostella

The β‐adrenergic‐like octopamine receptor (OA2B2) belongs to the class of G‐protein coupled receptors. It regulates important physiological functions in insects, thus is potentially a good target for insecticides. In this study, the putative open reading frame sequence of the Pxoa2b2 gene in Plutella xylostella was cloned. Orthologous sequence alignment, phylogenetic tree analysis, and protein sequence analysis all showed that the cloned receptor belongs to the OA2B2 protein family. PxOA2B2 was transiently expressed in HEK‐293 cells. It was found that PxOA2B2 could be activated by both octopamine and tyramine, resulting in increased intracellular cyclic AMP (cAMP) levels, whereas dopamine and serotonin were not effective in eliciting cAMP production. Further studies with series of PxOA2B2 agonists and antagonists showed that all four tested agonists (e.g., naphazoline, clonidine, 2‐phenylethylamine, and amitraz) could activate the PxOA2B2 receptor, and two of tested antagonists (e.g., phentolamine and mianserin) had significant antagonistic effects. However, antagonist of yohimbine had no effects. Quantitative real‐time polymerase chain reaction analysis showed that Pxoa2b2 gene was expressed in all developmental stages of P. xylostella and that the highest expression occurred in male adults. Further analysis with fourth‐instar P. xylostella larvae showed that the Pxoa2b2 gene was mainly expressed in Malpighian tubule, epidermal, and head tissues. This study provides both a pharmacological characterization and the gene expression patterns of the OA2B2 in P. xylostella, facilitating further research for insecticides using PxOA2B2 as a target.