Transcription Start Point Research Articles

Molecular mechanism of SugR-mediated sugar-dependent expression of the ldhA gene encoding l-lactate dehydrogenase in Corynebacterium glutamicum

This paper reports on the transcriptional regulation mechanism of the Corynebacterium glutamicum ldhA gene encoding L: -lactate dehydrogenase responsible for production of L: -lactate. DNA affinity purification allowed us to identify SugR, a global repressor of genes involved in sugar uptake and glycolysis, as a protein binding to the ldhA promoter region. Whereas ldhA gene expression and ldhA promoter activity were completely repressed during growth of wild-type cells in the absence of sugar, no such repression was observed in sugR mutant cells, indicating that SugR represses ldhA transcription. Electrophoretic mobility shift assays and DNase I footprint analyses revealed that two direct repeats, centered at position-17 with respect to the transcriptional start point, are required for SugR binding to the ldhA promoter and that fructose-1-phosphate (F-1-P) is the strongest negative effector of repressor activity of SugR. Furthermore, the ldhA promoter activity during growth on either fructose or sucrose, under which F-1-P is generated, is higher than on glucose, supporting the results of the in vitro binding assays. Thus, C. glutamicum ldhA is repressed by SugR in the absence of sugar and the expression level is dependent on the extent of derepression, which varies in response to sugars provided.

PolR, a pathway-specific transcriptional regulatory gene, positively controls polyoxin biosynthesis in Streptomyces cacaoi subsp. asoensis

The polyoxin (POL) biosynthetic gene cluster (pol) was recently cloned from Streptomyces cacaoi subsp. asoensis. A 3.3 kb DNA fragment carrying an obvious open reading frame (polR), whose deduced product shows sequence similarity to SanG of Streptomyces ansochromogenes and PimR of Streptomyces natalensis, was revealed within the pol gene cluster. Disruption of polR abolished POL production, which could be complemented by the integration of a single copy of polR into the chromosome of the non-producing mutant. The introduction of an extra copy of polR in the wild-type strain resulted in increased production of POLs. The transcription start point (tsp) of polR was determined by S1 mapping. Reverse transcriptase PCR experiments showed that PolR is required for the transcription of 18 structural genes in the pol gene cluster. Furthermore, we showed that polC and polB, the respective first genes of two putative operons (polC-polQ2 and polA-polB) consisting of 16 and 2 of these 18 genes, have similar promoter structures. Gel retardation assays indicated that PolR has specific DNA-binding activity for the promoter regions of polC and polB. Our data suggest that PolR acts in a positive manner to regulate POL production by activating the transcription of at least two putative operons in the pol gene cluster.

Transcriptional analysis of the gdhA gene in Streptococcus thermophilus

To study the transcriptional analysis of glutamate dehydrogenase gene, involved in the amino acid conversion to aroma compound in Streptococcus thermophilus. Analysis of the gdhA gene nucleotide sequence of S. thermophilus CNRZ1066 revealed that the coding region is 1353 nucleotides long. The deduced amino acids sequence exhibits the putative GDH active site and some conserved domains characteristic of family I of hexameric GDHs. Phylogenetic analysis revealed that the gdh gene of S. thermophilus clustered with the orthologues of other streptococci such as Streptococcus mutans, Streptococcus agalactiae and Streptococcus infantarius. Studying the structural organization of the gdhA locus the amino acid similarity of GDHs was higher than 87%, but the locus organization was not conserved. A dominant transcript of approximately 1.4 kbp was revealed by Northern blot hybridization, suggesting that gdhA mRNA is monocystronic. Primer extension showed that transcription start point of gdhA was localized 43 bp upstream of the potential start codon (ATG). The gdhA represents a monocistronic operon highly conserved in phylogenetic-related bacteria. A deeper knowledge of gdh transcriptional mechanisms could lead to develop S. thermophilus industrial starter cultures with optimized aromatic properties.

Dissection of phenotype reveals possible association between schizophrenia and Glutamate Receptor Delta 1 ( GRID1) gene promoter

Recent linkage and association data have implicated the Glutamate Receptor Delta 1 ( GRID1) locus in the etiology of schizophrenia. In this study, we sought to test whether variants in the promoter region are associated with this disorder. The distribution of CpG islands, which are known to be relevant for transcriptional regulation, was computationally determined at the GRID1 locus, and the putative transcriptional regulatory region at the 5′-terminus was systematically tagged using HapMap data. Genotype analyses were performed with 22 haplotype-tagging single nucleotide polymorphisms (htSNPs) in a German sample of 919 schizophrenia patients and 773 controls. The study also included two SNPs in intron 2 and one in intron 3 which have been found to be significantly associated with schizophrenia in previous studies. For the transcriptional regulatory region, association was obtained with rs3814614 ( p = 0.0193), rs10749535 ( p = 0.0245), and rs11201985 ( p = 0.0222). For all further analyses, the patient samples were divided into more homogeneous subgroups according to sex, age at onset, positive family history of schizophrenia and lifetime history of major depression. The p-value of the schizophrenia association finding for the three markers decreased by approximately one order of magnitude, despite the reduction in the total sample size. Marker rs3814614 (unadjusted p = 0.0005), located ~ 2.0 kb from the transcriptional start point, also withstood a two-step correction for multiple testing ( p = 0.030). No support was obtained for previously reported associations with the intronic markers. Our results suggest that genetic variants in the GRID1 transcriptional regulatory region may play a role in the etiology of schizophrenia, and that future association studies of schizophrenia may require stratification to ensure more homogeneous patient subgroups.

Diversity and transcription of proteases involved in the maturation of hydrogenases in Nostoc punctiforme ATCC 29133 and Nostocsp. strain PCC 7120

BackgroundThe last step in the maturation process of the large subunit of [NiFe]-hydrogenases is a proteolytic cleavage of the C-terminal by a hydrogenase specific protease. Contrary to other accessory proteins these hydrogenase proteases are believed to be specific whereby one type of hydrogenases specific protease only cleaves one type of hydrogenase. In cyanobacteria this is achieved by the gene product of either hupW or hoxW, specific for the uptake or the bidirectional hydrogenase respectively. The filamentous cyanobacteria Nostoc punctiforme ATCC 29133 and Nostoc sp strain PCC 7120 may contain a single uptake hydrogenase or both an uptake and a bidirectional hydrogenase respectively.ResultsIn order to examine these proteases in cyanobacteria, transcriptional analyses were performed of hupW in Nostoc punctiforme ATCC 29133 and hupW and hoxW in Nostoc sp. strain PCC 7120. These studies revealed numerous transcriptional start points together with putative binding sites for NtcA (hupW) and LexA (hoxW). In order to investigate the diversity and specificity among hydrogeanse specific proteases we constructed a phylogenetic tree which revealed several subgroups that showed a striking resemblance to the subgroups previously described for [NiFe]-hydrogenases. Additionally the proteases specificity was also addressed by amino acid sequence analysis and protein-protein docking experiments with 3D-models derived from bioinformatic studies. These studies revealed a so called "HOXBOX"; an amino acid sequence specific for protease of Hox-type which might be involved in docking with the large subunit of the hydrogenase.ConclusionOur findings suggest that the hydrogenase specific proteases are under similar regulatory control as the hydrogenases they cleave. The result from the phylogenetic study also indicates that the hydrogenase and the protease have co-evolved since ancient time and suggests that at least one major horizontal gene transfer has occurred. This co-evolution could be the result of a close interaction between the protease and the large subunit of the [NiFe]-hydrogenases, a theory supported by protein-protein docking experiments performed with 3D-models. Finally we present data that may explain the specificity seen among hydrogenase specific proteases, the so called "HOXBOX"; an amino acid sequence specific for proteases of Hox-type. This opens the door for more detailed studies of the specificity found among hydrogenase specific proteases and the structural properties behind it.

Characterization of the hupSL promoter activity in Nostoc punctiformeATCC 29133

BackgroundIn cyanobacteria three enzymes are directly involved in the hydrogen metabolism; a nitrogenase that produces molecular hydrogen, H2, as a by-product of nitrogen fixation, an uptake hydrogenase that recaptures H2 and oxidize it, and a bidirectional hydrogenase that can both oxidize and produce H2.Nostoc punctiforme ATCC 29133 is a filamentous dinitrogen fixing cyanobacterium containing a nitrogenase and an uptake hydrogenase but no bidirectional hydrogenase. Generally, little is known about the transcriptional regulation of the cyanobacterial uptake hydrogenases. In this study gel shift assays showed that NtcA has a specific affinity to a region of the hupSL promoter containing a predicted NtcA binding site. The predicted NtcA binding site is centred at 258.5 bp upstream the transcription start point (tsp). To further investigate the hupSL promoter, truncated versions of the hupSL promoter were fused to either gfp or luxAB, encoding the reporter proteins Green Fluorescent Protein and Luciferase, respectively.ResultsInterestingly, all hupsSL promoter deletion constructs showed heterocyst specific expression. Unexpectedly the shortest promoter fragment, a fragment covering 57 bp upstream and 258 bp downstream the tsp, exhibited the highest promoter activity. Deletion of the NtcA binding site neither affected the expression to any larger extent nor the heterocyst specificity.ConclusionObtained data suggest that the hupSL promoter in N. punctiforme is not strictly dependent on the upstream NtcA cis element and that the shortest promoter fragment (-57 to tsp) is enough for a high and heterocyst specific expression of hupSL. This is highly interesting because it indicates that the information that determines heterocyst specific gene expression might be confined to this short sequence or in the downstream untranslated leader sequence.

A motif within a complex promoter from the oomycete Phytophthora infestans determines transcription during an intermediate stage of sporulation

Sporulation in Phytophthora infestans is associated with a major remodeling of the transcriptome. To better understand promoter structure and how sporulation-specific expression is determined in this organism, the Pks1 gene was analyzed. Pks1 encodes a protein kinase that is induced at an intermediate stage of sporulation, prior to sporangium maturation. Major and minor transcription start sites mapped throughout the promoter, which contains many T-rich stretches and Inr-like elements. Within the T-rich region are several motifs which bound nuclear proteins in EMSA. Tests of modified promoters in transformants implicated a CCGTTG located 110-nt upstream of the transcription start point as a major regulator of sporulation-specific transcription. The motif also bound a sporulation-specific nuclear protein complex. A bioinformatics analysis indicated that the motif is highly over-represented within co-expressed promoters, in which it predominantly resides 100–300-nt upstream of transcription start sites. Other sequences, such as a CATTTGTT motif, also bound nuclear proteins but did not play an essential role in spore-specific expression.

Negative modulation of the chicken infectious anemia virus promoter by COUP-TF1 and an E box-like element at the transcription start site binding δEF1

Expression of enhanced green fluorescent protein (EGFP) under control of the promoter-enhancer of chicken infectious anemia virus (CAV) is increased in an oestrogen receptor-enhanced cell line when treated with oestrogen and the promoter-enhancer binds unidentified proteins that recognize a consensus oestrogen response element (ERE). Co-transfection assays with the CAV promoter and the nuclear receptor chicken ovalbumin upstream promoter transcription factor 1 (COUP-TF1) showed that expression of EGFP was decreased by 50 to 60 % in DF-1 and LMH cells. The CAV promoter that included sequences at and downstream of the transcription start point had less expression than a short promoter construct. Mutation of a putative E box at this site restored expression levels. Electromobility shift assays showed that the transcription regulator delta-EF1 (deltaEF1) binds to this E box region. These findings indicate that the CAV promoter activity can be affected directly or indirectly by COUP-TF1 and deltaEF1.

Expression of Mycobacterium tuberculosis pe_pgrs33 is repressed during stationary phase and stress conditions, and its transcription is mediated by sigma factor A

Although recent work shows that the expression of the PE/PE_PGRS protein family occur both in vitro and in vivo under stress conditions, very little is known about their promoter and how they are regulated. In this work, the promoter region of a member of PE_PGRS family, the PE_PGRS33 was identified and the promoter boxes were determined. To date, this is one of the few reports that describe a promoter region of a PE_PGRS member. In addition, the gene promoter functionality was assayed in Mycobacterium smegmatis with the green fluorescent protein reporter gene fused to different lengths of pe_pgrs33 promoter sequences. The GFP was down-regulated in the stationary phase, under nutrient starvation and oxygen depletion, suggesting that, in stress conditions, regulation of the gene could be under control of a repressor molecule. A 5′ rapid amplification of cDNA end assay of transcriptional fusions evaluated in M. smegmatis and in Mycobacterium tuberculosis mRNA revealed a transcription start point 75 nt upstream of the ATG codon and a −10 like-SigA box. Furthermore, a transcription run assay confirmed that SigA mediates in vitro transcription of pe_pgrs33. Interestingly, conserved −10 SigA boxes were found in the intergenic region of several PE_PGRS genes. These results suggest that expression of some PE_PGRS genes may be mediated by SigA, and the differences in expression observed in the gene family could be explained by the participation of additional regulatory genetic elements.

Promoter Analysis of Palindromic Transcription Units in the Ribosomal DNA Circle of Entamoeba histolytica

rRNA genes of Entamoeba histolytica are organized as palindromic ribosomal DNA (rDNA) units (I and II) in a 24.5-kb circle. Although the two rDNAs are identical in sequence, their upstream spacers are completely different. Since the intergenic sequences (IGS) of all rDNA copies in other organisms are conserved and contain transcription regulatory sequences, the lack of sequence conservation in the IGS prompted the question of whether both rDNAs are indeed transcriptionally active. We mapped the transcriptional start points (tsp's) and promoters of the two rDNAs. A 51-bp sequence immediately upstream of the tsp's was highly conserved in both units. In addition, both units had an A+T-rich stretch upstream of the 51-bp core. Analysis of reporter gene transcription showed promoter activity to reside in the regions from positions -86 to +123 (rDNA I) and positions -101 to +140 (rDNA II). The promoter-containing fragments from both units could bind and compete with each other for protein(s) from nuclear extracts. Protein binding was especially dependent on the A+T-rich region upstream of the 51-bp core (positions -53 to -68). The requirement of >80 bp downstream of the tsp was striking. Although this sequence was not conserved in the two units, it could potentially fold into very long stem-loops. Both rDNAs transcribed with comparable efficiency, as measured by nuclear runon. Thus, both rDNAs share very similar organization of promoter sequences, and in exponential culture both rDNAs are transcribed. It remains to be seen whether the different IGS affect the regulation of the two units under adverse conditions.

The dual transcriptional regulator CysR in Corynebacterium glutamicum ATCC 13032 controls a subset of genes of the McbR regulon in response to the availability of sulphide acceptor molecules

BackgroundRegulation of sulphur metabolism in Corynebacterium glutamicum ATCC 13032 has been studied intensively in the last few years, due to its industrial as well as scientific importance. Previously, the gene cg0156 was shown to belong to the regulon of McbR, a global transcriptional repressor of sulphur metabolism in C. glutamicum. This gene encodes a putative ROK-type regulator, a paralogue of the activator of sulphonate utilisation, SsuR. Therefore, it is an interesting candidate for study to further the understanding of the regulation of sulphur metabolism in C. glutamicum.ResultsDeletion of cg0156, now designated cysR, results in the inability of the mutant to utilise sulphate and aliphatic sulphonates. DNA microarray hybridisations revealed 49 genes with significantly increased and 48 with decreased transcript levels in presence of the native CysR compared to a cysR deletion mutant. Among the genes positively controlled by CysR were the gene cluster involved in sulphate reduction, fpr2 cysIXHDNYZ, and ssuR. Gel retardation experiments demonstrated that binding of CysR to DNA depends in vitro on the presence of either O-acetyl-L-serine or O-acetyl-L-homoserine. Mapping of the transcription start points of five transcription units helped to identify a 10 bp inverted repeat as the possible CysR binding site. Subsequent in vivo tests proved this motif to be necessary for CysR-dependent transcriptional regulation.ConclusionCysR acts as the functional analogue of the unrelated LysR-type regulator CysB from Escherichia coli, controlling sulphide production in response to acceptor availability. In both bacteria, gene duplication events seem to have taken place which resulted in the evolution of dedicated regulators for the control of sulphonate utilisation. The striking convergent evolution of network topology indicates the strong selective pressure to control the metabolism of the essential but often toxic sulphur-containing (bio-)molecules.

HetF and PatA Control Levels of HetR in Anabaena sp. Strain PCC 7120

Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium that differentiates heterocysts in response to deprivation of combined nitrogen. A hetF deletion strain lacked heterocysts and had aberrant cell morphology. Site-directed mutagenesis of the predicted active-site histidine and cysteine residues of this putative caspase-hemoglobinase fold protease abolished HetF function, supporting the hypothesis that HetF is a protease. Deletion of patA, which is necessary for the formation of most intercalary heterocysts, or hetF resulted in an increase in HetR protein, and extra copies of hetF on a plasmid functionally bypassed the deletion of patA. A hetR-gfp translational fusion expressed from an inducible promoter demonstrated that hetF-dependent downregulation of HetR levels occurs rapidly in vegetative cells, as well as developing heterocysts. "Mosaic" filaments in which only one cell of a filament had a copy of hetR or hetF indicated that hetF is required for differentiation only in cells that will become heterocysts. hetF was required for transcription from a hetR-dependent transcription start point of the hetR promoter and induction of transcription from the patS promoter. The inverse correlation between the level of HetR protein and transcription from hetR-dependent promoters suggests that the transcriptional activity of HetR is regulated by HetF and PatA.

Characterization of the Aspergillus niger prtT, a unique regulator of extracellular protease encoding genes

Expression of several Aspergillus niger genes encoding major secreted, but not vacuolar, protease genes including the major acid protease gene pepA, was shown to be affected in the previously isolated A. niger protease mutant, AB1.13 [Mattern, I.E., van Noort, J.M., van den Berg, P., Archer, D.A., Roberts, I.N., Hondel, C.A.M.J.J., 1992. Isolation and characterization of mutants of Aspergillus niger deficient in extracellular proteases. Molecular & General Genetics 2, 332–336]. Complementation cloning of the putative protease-regulatory gene affected in this mutant was accomplished using a functional selection approach based on the use of the A. nidulans amdS selection marker driven by the A. niger pepA promoter. As expected the PpepA::amdS selection marker is not expressed in the mutant. Introduction of a self-replicating cosmid library into the mutant strain carrying the PpepA::amdS marker allowed selection of AmdS+ transformants functionally complementing the proposed regulatory mutation. Analysis of complementing cosmid clones revealed that the complementing sequences contained a gene encoding a member of the fungal-specific Zn2Cys6-binuclear cluster protein family. Sequence comparison of the encoded protein, PrtT, showed that it has homologues among different Aspergillus species. The A. oryzae homologue was shown to govern expression of the major alkaline protease AlpA and neutral protease Np1 in this species. In contrast to several other pathway specific regulators, such as AmyR and XlnR, no PrtT orthologues could be found in any other non- Aspergillus (or related) species and surprisingly, also not in Aspergillus nidulans. Interestingly, in all Aspergillus species carrying a prtT orthologue the gene is tightly clustered to a completely syntenous region carrying an amylolytic gene cluster including another Zn2Cys6-binuclear cluster protein, AmyR. Northern analysis of the A. niger prtT gene showed (constitutive) expression from two upstream promoters about 700 bp apart. The presence of several short upstream open reading frames downstream of both the distal and the proximal transcription start point of the prtT gene suggests regulation at the post-translational level. Also regulation at the level of differential splicing is suggested by the fact that several Aspergillus EST databases carry a considerable fraction of clones in which in frame intron sequences are retained.

Energetic contributions to the initiation of transcription in E. coli

The thermodynamics of RNA polymerase (RNAP) binding to a 108 base pair (bp) synthetic promoter with consensus sequences at the − 35 and − 10 bp binding regions upstream from the transcription start point were determined using isothermal titration calorimetry (ITC). The binding constant at 25 °C is 2.37 ± 0.18 × 10 7 M − 1 , which is reduced to 0.17 ± 0.06 × 10 7 M − 1 with mutations in the − 10 bp region but remained the same with mutations in the − 35 binding region. The binding reactions were enthalpically-driven with exothermic binding enthalpies ranging from − 57 ± 6 kJ mol − 1 at 15 °C to − 271 ± 20 kJ mol − 1 at 35 °C yielding a large binding heat capacity change of − 10.7 ± 1.9 kJ mol − 1 K − 1 , indicating a conformational change upon binding to the RNAP. Differential scanning calorimetry (DSC) scans of the thermal unfolding of RNAP and the promoter–RNAP complex exhibited an unfolding transition at 55.5 ± 0.6 °C and at 58.9 ± 0.5 °C for the RNAP but only one transition at 60.5 ± 1.1 °C for the complex with van't Hoff enthalpy to transition enthalpy ratios of, resp., 3.2 ± 0.3 and 4.3 ± 0.5. The single transition of the complex results from a shift to 60.5 °C of the low temperature transition upon promoter binding to the structural unit unfolding at the lower temperature in RNAP. The large transition enthalpy ratios indicate that the σ, α, α, β, and β′ subunits unfold as almost independent entities. The dissociation thermodynamics of short transcription “bubble” duplexes of 7 promoters sequenced from − 1 to − 12 bp were determined from ITC and DSC measurements. The free energy change of the promoter binding to the RNAP and the free energy requirement for formation of the transcription bubble at the low promoter concentrations in the cell are sufficient to drive the initiation of transcription through the isomerization of the closed to the open form step of the RNAP–promoter complex.

Iron availability affects mcyD expression and microcystin‐LR synthesis in Microcystis aeruginosa PCC7806

Microcystins are toxins produced by cyanobacteria that entail serious health and environmental problems. They are cyclic heptapeptides synthesized via a mixed polyketide synthase/non-ribosomal peptide synthetase system called microcystin synthetase. Environmental and nutritional factors that trigger microcystin synthesis are still debated and this work deals with the study of the influence of iron nutritional status on the microcystin synthesis. The results indicate that iron deficiency could be one of the inducing factors of the microcystin synthesis. For the first time, increased transcription of an essential mcy gene and correlative microcystin synthesis has been established. Real-time PCR analysis of mcyD, and microcystin-LR synthesis were studied on Microcystis aeruginosa PCC7806 grown in iron-replete and iron-deplete media. Iron starvation causes an increase of mcyD transcription, correlative to the increase of microcystin-LR levels. Four transcription start points were identified for mcyD and two for mcyA, and they are not changed as a consequence of iron deficiency.

Mutant rat strain lacking d-amino-acid oxidase

D-amino-acid oxidase (DAO) is known to be associated with schizophrenia. Since the expression of DAO gene had been reported to be very low in LEA rats, we examined LEA/SENDAI rats in detail. These rats did not have DAO activity, enzyme protein or mRNA encoding this enzyme. Sequencing of the 5'-upstream region of the DAO gene revealed the deletion of one triplet in the 15 TAA repeats approximately 700-bp upstream of the transcription start point. A 1.3-kb upstream fragment containing the TAA repeats and the transcription start point was inserted into a reporter vector and was transfected into COS-1, NRK-52E and CCL-PK1 cells. Although the fragments containing 15 or 14 repeats had high promoter activity, the fragment containing 13 repeats had very weak activity. Electrophoretic mobility-shift assays showed that the nuclear extracts from COS-1 and COS-7 cells had proteins that bound to the oligonucleotides containing the TAA repeats. These results suggest that the TAA repeats are important for expression of the DAO gene. The LEA/SENDAI rats lacking DAO would be a useful tool for the investigations aimed at the elucidation of the relationships between this flavoenzyme and schizophrenia.

The Two-Component System PhoPR of Clostridium acetobutylicum Is Involved in Phosphate-Dependent Gene Regulation

The phoPR gene locus of Clostridium acetobutylicum ATCC 824 comprises two genes, phoP and phoR. Deduced proteins are predicted to represent a response regulator and sensor kinase of a phosphate-dependent two-component regulatory system. We analyzed the expression patterns of phoPR in P(i)-limited chemostat cultures and in response to P(i) pulses. A basic transcription level under high-phosphate conditions was shown, and a significant increase in mRNA transcript levels was found when external P(i) concentrations dropped below 0.3 mM. In two-dimensional gel electrophoresis experiments, a 2.5-fold increase in PhoP was observed under P(i)-limiting growth conditions compared to growth with an excess of P(i). At least three different transcription start points for phoP were determined by primer extension analyses. Proteins PhoP and an N-terminally truncated *PhoR were individually expressed heterologously in Escherichia coli and purified. Autophosphorylation of *PhoR and phosphorylation of PhoP were shown in vitro. Electromobility shift assays proved that there was a specific binding of PhoP to the promoter region of the phosphate-regulated pst operon of C. acetobutylicum.

Phosphate-dependent regulation of the low- and high-affinity transport systems in the model actinomycete Streptomyces coelicolor

The transport of inorganic phosphate (P(i)) is essential for the growth of all organisms. The metabolism of soil-dwelling Streptomyces species, and their ability to produce antibiotics and other secondary metabolites, are strongly influenced by the availability of phosphate. The transcriptional regulation of the SCO4138 and SCO1845 genes of Streptomyces coelicolor was studied. These genes encode the two putative low-affinity P(i) transporters PitH1 and PitH2, respectively. Expression of these genes and that of the high-affinity transport system pstSCAB follows a sequential pattern in response to phosphate deprivation, as shown by coupling their promoters to a luciferase reporter gene. Expression of pitH2, but not that of pap-pitH1 (a bicistronic transcript), is dependent upon the response regulator PhoP. PhoP binds to specific sequences consisting of direct repeats of 11 nt in the promoter of pitH2, but does not bind to the pap-pitH1 promoter, which lacks these direct repeats for PhoP recognition. The transcription start point of the pitH2 promoter was identified by primer extension analyses, and the structure of the regulatory sequences in the PhoP-protected DNA region was established. It consists of four central direct repeats flanked by two other less conserved repeats. A model for PhoP regulation of this promoter is proposed based on the four promoter DNA-PhoP complexes detected by electrophoretic mobility shift assays and footprinting studies.

The plant-specific TFIIB-related protein, pBrp, is a general transcription factor for RNA polymerase I

TFIIB and BRF are general transcription factors (GTFs) for eukaryotic RNA polymerases II and III, respectively, and have important functions in transcriptional initiation. In this study, the third type of TFIIB-related protein, pBrp, found in plant lineages was characterized in the red alga Cyanidioschyzon merolae. Chromatin immunoprecipitation analysis revealed that CmpBrp specifically occupied the rDNA promoter region in vivo, and the occupancy was proportional to de novo 18S rRNA synthesis. Consistently, CmpBrp and CmTBP cooperatively bound the rDNA promoter region in vitro, and the binding site was identified at a proximal downstream region of the transcription start point. alpha-Amanitin-resistant transcription from the rDNA promoter in crude cell lysate was severely inhibited by the CmpBrp antibody and was also inhibited when DNA template with a mutated CmpBrp-CmTBP binding site was used. CmpBrp was shown to co-immunoprecipitate and co-localize with the RNA polymerase I subunit, CmRPA190, in the cell. Thus, together with comparative studies of Arabidopsis pBrp, we concluded that pBrp is a GTF for RNA polymerase I in plant cells.

The interplay of StyR and IHF regulates substrate-dependent induction and carbon catabolite repression of styrene catabolism genes in Pseudomonas fluorescens ST

BackgroundIn Pseudomonas fluorescens ST, the promoter of the styrene catabolic operon, PstyA, is induced by styrene and is subject to catabolite repression. PstyA regulation relies on the StyS/StyR two-component system and on the IHF global regulator. The phosphorylated response regulator StyR (StyR-P) activates PstyA in inducing conditions when it binds to the high-affinity site STY2, located about -40 bp from the transcription start point. A cis-acting element upstream of STY2, named URE, contains a low-affinity StyR-P binding site (STY1), overlapping the IHF binding site. Deletion of the URE led to a decrease of promoter activity in inducing conditions and to a partial release of catabolite repression. This study was undertaken to assess the relative role played by IHF and StyR-P on the URE, and to clarify if PstyA catabolite repression could rely on the interplay of these regulators.ResultsStyR-P and IHF compete for binding to the URE region. PstyA full activity in inducing conditions is achieved when StyR-P and IHF bind to site STY2 and to the URE, respectively. Under catabolite repression conditions, StyR-P binds the STY1 site, replacing IHF at the URE region. StyR-P bound to both STY1 and STY2 sites oligomerizes, likely promoting the formation of a DNA loop that closes the promoter in a repressed conformation. We found that StyR and IHF protein levels did not change in catabolite repression conditions, implying that PstyA repression is achieved through an increase in the StyR-P/StyR ratio.ConclusionWe propose a model according to which the activity of the PstyA promoter is determined by conformational changes. An open conformation is operative in inducing conditions when StyR-P is bound to STY2 site and IHF to the URE. Under catabolite repression conditions StyR-P cellular levels would increase, displacing IHF from the URE and closing the promoter in a repressed conformation. The balance between the open and the closed promoter conformation would determine a fine modulation of the promoter activity. Since StyR and IHF protein levels do not vary in the different conditions, the key-factor regulating PstyA catabolite repression is likely the kinase activity of the StyR-cognate sensor protein StyS.