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We have examined the effects on transcription initiation of promoter and enhancer strength and of the curvature of the DNA separating these entities on wild-type and mutated enhancer-promoter regions at the Escherichia coli sigma54-dependent promoters glnAp2 and glnHp2 on supercoiled and linear DNA. Our results, together with previously reported ob...
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... but not on linear, DNA; however, in this case, substitution of the higher affinity glnAp2 promoter for nifLp, resulting in a hybrid template with the nifL enhancer and intervening region fused to glnAp2, allowed transcription to be initiated on linear DNA (17,18). Our computer analysis of the nifF nifL enhancer- promoter region, illustrated in Fig. 6, shows that the DNA is bent in the center of the region, with enhancers and promoters in the same plane and all bound proteins located on the same face of the DNA helix. The inability of the nifLp promoter to support the initiation of transcription on linear DNA is therefore not due to the lack of intrinsic curvature of the DNA ...
Citations
... One possibility is that the inhibitory amino acid-induced glutamine limitation results in guanosine tetraphosphate accumulation, which has been recently shown to repress transcription of the DNA gyrase genes (19). Because transcrip tion from glnAp2 requires supercoiled DNA (20)(21)(22), the resulting supercoiling relaxation may be sufficient to activate glnAp1-dependent glnA transcription. Several amino acids have been known to inhibit GS activity by binding to the glutamate substrate (17,23,24). ...
Growth of uropathogenic Escherichia coli in the bladder induces transcription of glnA which codes for the ammonia-assimilating glutamine synthetase (GS) despite the normally suppressive high ammonia concentration. We previously showed that the major urinary component, urea, induces transcription from the Crp-dependent glnAp1 promoter, but the urea-induced transcript is not translated. Our purpose here was to determine whether the most abundant urinary amino acids, which are known to inhibit GS activity in vitro, also affect glnA transcription in vivo. We found that the abundant amino acids impaired growth, which glutamine and glutamate reversed; this implies inhibition of GS activity. In strains with deletions of crp and glnG that force transcription from the glnAp2 and glnAp1 promoters, respectively, we examined growth and glnA transcription with a glnA-gfp transcriptional fusion and quantitative reverse transcription PCR with primers that can distinguish transcription from the two promoters. The abundant urinary amino acids stimulated transcription from the glnAp2 promoter in the absence of urea but from the glnAp1 promoter in the presence of urea. However, transcription from glnAp1 did not produce a translatable mRNA or GS as assessed by a glnA-gfp translational fusion, enzymatic assay of GS, and Western blot to detect GS antigen in urea-containing media. We discuss these results within the context of the extremely rapid growth of uropathogenic E. coli in urine, the different factors that control the two glnA promoters and possible mechanisms that either overcome or bypass the urea-imposed block of glutamine synthesis during bacterial growth in urine.
IMPORTANCE
Knowledge of the regulatory mechanisms for genes expressed at the site of infection provides insight into the virulence of pathogenic bacteria. During urinary tract infections—most often caused by Escherichia coli—growth in urine induces the glnA gene which codes for glutamine synthetase. The most abundant urinary amino acids amplified the effect of urea which resulted in hypertranscription from the glnAp1 promoter and, unexpectedly, an untranslated transcript. E. coli must overcome this block in glutamine synthesis during growth in urine, and the mechanism of glutamine acquisition or synthesis may suggest a possible therapy.
... We need not consider whether there exist other NtrC oligomeric structures since NtrC is recruited in units of a dimer and only hexamers catalyze the holoenzyme (11,38). We also ignore the rather small affinity difference (58). It separately takes ∼64 s and 80 s on average for a hexamer at enhancer I or II to encounter the holoenzyme. ...
Transcription initiation is orchestrated by dynamic molecular interactions, with kinetic steps difficult to detect. Utilizing a hybrid method, we aim to unravel essential kinetic steps of transcriptional regulation on the glnAp2 promoter, whose regulatory region includes two enhancers (sites I and II) and three low-affinity sequences (sites III-V), to which the transcriptional activator NtrC binds. By structure reconstruction, we analyze all possible organization architectures of the transcription apparatus (TA). The main regulatory mode involves two NtrC hexamers: one at enhancer II transiently associates with site V such that the other at enhancer I can rapidly approach and catalyze the σ(54)-RNA polymerase holoenzyme. We build a kinetic model characterizing essential steps of the TA operation; with the known kinetics of the holoenzyme interacting with DNA, this model enables the kinetics beyond technical detection to be determined by fitting the input-output function of the wild-type promoter. The model further quantitatively reproduces transcriptional activities of various mutated promoters. These results reveal different roles played by two enhancers and interpret why the low-affinity elements conditionally enhance or repress transcription. This work presents an integrated dynamic picture of regulated transcription initiation and suggests an evolutionarily conserved characteristic guaranteeing reliable transcriptional response to regulatory signals.
... Способность геномной ДНК быть матрицей для синтеза РНК в значительной степени определяется структурно-конформационным состоянием её регуляторных участков (промоторов) [1][2][3][4][5][6][7][8]. Первостепенную роль в создании оптимальной пространственной конфигурации промоторной ДНК играют разнообразные белковые факторы, которые в зависимости от потребности способствуют или, наоборот, препятствуют формированию транскрипционного комплекса. ...
... Several groups (Carmona et al. 1997 ;Cheema et al. 1999) reported an apparent effect of the presence of intrinsically bent regions (containing A-tracts) on transcription activation in promoters that do not contain binding sites for proteins, such as IHF, that bend DNA. Cheema et al. (1999) studied transcription activation from the nifLA promoter of Klebsiella pneumoniae. ...
... However, later studies suggested that these effects may be due to the deletion or alteration of cryptic subsidiary NtrC binding sites (Lilja et al. 2004). The glnAp2 promoter of E. coli and Salmonella typhinum is a prototype for looping mediate transcription activation (Rombel et al. 1998) and supercoiling has been shown to be important for transcription activation in glnAp2 (Carmona et al. 1997 ;Liu et al. 2001). Schulz et al. (2000) studied transcription from the glnAp2 promoter and reaffirmed the importance of supercoiling for transcription activation in this system. ...
Short runs of adenines are a ubiquitous DNA element in regulatory regions of many organisms. When runs of 4-6 adenine base pairs ('A-tracts') are repeated with the helical periodicity, they give rise to global curvature of the DNA double helix, which can be macroscopically characterized by anomalously slow migration on polyacrylamide gels. The molecular structure of these DNA tracts is unusual and distinct from that of canonical B-DNA. We review here our current knowledge about the molecular details of A-tract structure and its interaction with sequences flanking them of either side and with the environment. Various molecular models were proposed to describe A-tract structure and how it causes global deflection of the DNA helical axis. We review old and recent findings that enable us to amalgamate the various findings to one model that conforms to the experimental data. Sequences containing phased repeats of A-tracts have from the very beginning been synonymous with global intrinsic DNA bending. In this review, we show that very often it is the unique structure of A-tracts that is at the basis of their widespread occurrence in regulatory regions of many organisms. Thus, the biological importance of A-tracts may often be residing in their distinct structure rather than in the global curvature that they induce on sequences containing them.
... This is further supported by the results for the hpxP transcription initiation site, which showed that in cells grown in hypoxanthine, hpxPQT transcription seems to be dependent on 54 . Furthermore, NtrC and IHF binding sites have been identified in the promoter region in locations compatible with NtrC-mediated control, as reported for other NtrC-controlled genetic systems (6,36,48). The fact that hpxPQT expression was not completely turned off in the rpoN mutant suggests that another promoter may drive its transcription. ...
Growth experiments showed that adenine and hypoxanthine can be used as nitrogen sources by several strains of K. pneumoniae under aerobic conditions. The assimilation of all nitrogens from these purines indicates that the catabolic pathway is complete
and proceeds past allantoin. Here we identify the genetic system responsible for the oxidation of hypoxanthine to allantoin
in K. pneumoniae. The hpx cluster consists of seven genes, for which an organization in four transcriptional units, hpxDE, hpxR, hpxO, and hpxPQT, is proposed. The proteins involved in the oxidation of hypoxanthine (HpxDE) or uric acid (HpxO) did not display any similarity
to other reported enzymes known to catalyze these reactions but instead are similar to oxygenases acting on aromatic compounds.
Expression of the hpx system is activated by nitrogen limitation and by the presence of specific substrates, with hpxDE and hpxPQT controlled by both signals. Nitrogen control of hpxPQT transcription, which depends on σ54, is mediated by the Ntr system. In contrast, neither NtrC nor the nitrogen assimilation control protein is involved in the
nitrogen control of hpxDE, which is dependent on σ70 for transcription. Activation of these operons by the specific substrates is also mediated by different effectors and regulatory
proteins. Induction of hpxPQT requires uric acid formation, whereas expression of hpxDE is induced by the presence of hypoxanthine through the regulatory protein HpxR. This LysR-type regulator binds to a TCTGC-N4-GCAAA site in the intergenic hpxD-hpxR region. When bound to this site for hpxDE activation, HpxR negatively controls its own transcription.
... RpoN binds to RNA polymerase to form a closed holoenzyme complex that requires further activation by members of the NtrC class of enhancer-binding proteins (EBPs) (Studholme and Buck, 2000;Studholme and Dixon, 2003). Transcriptional activation by RpoN requires nucleotide hydrolysis by EBPs, which typically bind promoters at a distance from the transcription start site and contact RpoN by DNA looping ( Carmona et al., 1997;Studholme and Buck, 2000;Studholme and Dixon, 2003). DNA looping and supercoiling may be enhanced by the DNA-bending activity of IHF ( Carmona et al., 1997;Dworkin et al., 1997;Palacios and EscalanteSemerena, 2000;Sze et al., 2001). ...
... Transcriptional activation by RpoN requires nucleotide hydrolysis by EBPs, which typically bind promoters at a distance from the transcription start site and contact RpoN by DNA looping ( Carmona et al., 1997;Studholme and Buck, 2000;Studholme and Dixon, 2003). DNA looping and supercoiling may be enhanced by the DNA-bending activity of IHF ( Carmona et al., 1997;Dworkin et al., 1997;Palacios and EscalanteSemerena, 2000;Sze et al., 2001). Environmental signal transduction occurs via activation or de-repression of EBPs, although general metabolic signals such as the stringent response signal (p)ppGpp and alterations in DNA supercoiling also affect a subset of RpoN-dependent promoters (Sze and Shingler, 1999;Carmona et al., 2000;Sze et al., 2001;Paul et al., 2005;Bernardo et al., 2006). ...
The alternative sigma factor RpoN is a key regulator in the acclimation of Pseudomonas to complex natural environments. In this study we show that RpoN is required for efficient colonization of sugar beet seedlings by the plant growth-promoting bacterium Pseudomonas fluorescens SBW25, and use phenotypic and bioinformatic approaches to profile the RpoN-dependent traits and genes of P. fluorescens SBW25. RpoN is required for flagellar biosynthesis and for assimilation of a wide variety of nutrient sources including inorganic nitrogen, amino acids, sugar alcohols and dicarboxylic acids. Chemosensitivity assays indicate that RpoN-regulated genes contribute to acid tolerance and resistance to some antibiotics, including tetracyclines and aminoglycosides. Gain of function changes associated with loss of RpoN included increased tolerance to hydroxyurea and Guanazole. Bioinformatic predictions of RpoN-regulated genes show a close correspondence with phenotypic analyses of RpoN-regulated traits and suggest novel functions for RpoN in P. fluorescens, including regulation of poly(A) polymerase. The reduced plant colonization ability observed for an rpoN mutant of P. fluorescens is therefore likely to be due to defects in multiple traits including nutrient assimilation, protein secretion and stress tolerance.
... ơ 54 regulated genes have a binding motif (GC/GG) in their promoter upstream sequences located at -12/-24 relative to the transcription start site and an additional enhancer sequence, usually situated between -100 to -150 (Carmona et al. 1997). ...
... They bind to DNA when phosphorylated at a specific site in their receiver domain (see section 1.7.2). Activators of this type interact with DNA and σ 54 at the same time to introduce a bend in DNA, ultimately forming an open promoter complex as a prerequisite for transcription (Carmona et al. 1997). ...
The Gram-negative soil bacterium Pseudomonad putida is able to utilize an unusually large number of organic compounds as sources of energy and biomass, among them the acidic amino acids glutamate (Glu) and aspartate (Asp) and their amides glutamine (Gln) and asparagine (Asn). During growth of P. putida on any of these amino acids, the expression of a defined set of genes is induced that allow for their utilization. As reported previously, the Aau two-component system is involved in this adaptation process. In the present study we analyzed the functional role of the AauR-AauS system in the uptake and metabolism of acidic amino acid by P. putida KT2440. Aau-negative mutants were defective in growth Glu and Asp as sole sources of carbon and nitrogen as well as in Glu and Asp uptake. In addition, the AauR mutant failed to express periplasmic glutaminase/ asparaginase (PGA), an enzyme required for the conversion of Gln and Asn to the respective acidic amino acids. Other enzymes involved in the assimilation of Glu and Asp also showed marked changes in activity. AauR deletion mutants grown on Asn and Asp started to accumulate Glu in the late log phase to levels many times higher than in the wild type and also excreted Glu if glucose was available. The excretion of glutamate in these conditions is probably mediated by Bra (a bi-directional ABC transporter), as reported for TCA cycle mutants of other rhizobacteria. The glutamate accumulation by aau mutants may be due to high aspartase activities which feed the carbon skeletons of Asp and Asn into the TCA cycle where glutamate dehydrogenase converts it to glutamate. Immediately upstream of the aau locus of P. putida KT2440, an operon involving genes PP1068-PP1071 encodes an ABC transporter which we named Aat (for acidic amino acid transporter). Aat is also upregulated when P. putida is grown on acidic amino acids or their amides. AatPMQJ is a polar amino acid transporter belonging to the solute-binding protein family (SBP_Bac_3). The Aat system consists of four subunits, where AatP is the nucleotide binding protein, AatM and Aat Q are membrane spanning permeases and AatJ is the periplasmic solute-binding protein. The expressed and purified solute binding protein (AatJ) showed high binding affinity towards Glu and Asp (Kd = 0.4 μM and 1.3 μM respectively), while Gln and Asn as well as other dicarboxylates were bound with much lower affinity. A modeled structure of AatJ (using the glutamine- binding protein GlnH of E. coli as template) suggested a novel arrangement of active site residues which include several arginine residues. The modeled structure was validated by site directed mutagenesis of several AatJ residues predicted to be in contact with the bound ligand. A data base search suggested that AatJ and at least 15 further proteins constitute a new subfamily of periplasmic acidic amino acid receptors. The role of the AauRS two-component system in the transcriptional regulation of aat and the PGA-encoding ansB gene was analyzed by gel-shift assays. The purified recombinant protein AauR was shown to bind to the promoter regions of both aat and ansB. A DNase I footprinting analysis revealed that the AauR binding motif consists of a well-conserved inverted repeat of six nucleotides (GTTCGGNNNNCCGAAC). By in-silico analysis of the P. putida KT2440 genome, several other genes were detected that contain an AauR interaction motif in their promoters. These genes encode a H+/Glu symporter (GltP), phosphoenolpyruvate synthase (PpsA), a branched-chain amino acid transporter (Bra) and a disulphide exchange protein (DsbC). Based on these data, we give an outline of acidic amino acid uptake and metabolism in P. putida and its regulation by the AauRS two-component system. Das Gram-negative Bodenbakterium Pseudomonas putida ist in der Lage, eine ungewöhnlich große Zahl organischer Verbindungen als Quelle für Energie und Biomasse zu nutzen. Unter diesen Verbindungen sind auch die sauren Aminosäuren Glutamat (Glu) und Aspartat (Asp) sowie deren Amide Glutamin (Gln) und Asparagin (Asn). Wächst P. putida auf einer dieser Verbindungen, wird eine Gruppe von Genen induziert, die für Aufnahme und Verwertung dieser Aminosäuren notwendig sind. Aus früheren Untersuchungen war bereits bekannt, dass das Zweikom¬¬po¬nentensystem AauRS an diesem Anpassungsprozess beteiligt ist. In der vorliegenden Arbeit untersuchten wir am Beispiel des Stammes P. putida KT2440 die Rolle des Aau-Systems bei der Aufnahme und Verstoffwechslung der sauren Aminosäuren im Einzelnen. Aau-negative Mutanten waren unfähig, auf Glu and Gln als einziger C- und N-Quelle zu wachsen und zeigten auch massive Defekte in der Aufnahme von Glu und Asp. Außerdem waren sie nicht mehr in der Lage, periplasmatische Glutaminase/Asparaginase (PGA) zu exprimieren, ein Enzym das die die Hydrolyse von Gln und Asn zu den betreffenden Dicarboxylaten katalysiert. Auch andere Enzyme des Glu- und Asp-Stoffwechsel waren in ihrer Aktivität verändert. Mutanten mit inaktiviertem AauR akkumulierten beim Wachstums auf Asn und Asp intrazellulär große Mengen von Glutamat. Die dabei auftretenden Glutamatspiegel waren um ein Vielfaches höher als in Wildtyp-Zellen. Diese Exkretion von Glutamat, die auch bei Citratzyklus-Mutanten anderer Rhizobakterien beobachtet wurde, wird wahrschein¬lich durch den bidrektionalen ABC-Transporter Bra bewirkt. Die Anhäufung von Glutamat geht vermutlich auf die in AauR-Mutanten erhöhten Aspartase-Aktivitäten zurück. Dieses Enzym kanalisiert das Kohlenstoffskelett von Asp in den Citratzyklus, von wo aus durch die Glutamatdehydrogenase Glutamat gebildet werden kann. Im Genom von P. putida KT2440 liegt direkt neben aau ein weiteres Operon, das von den Genen PP1068-PP1071 gebildet wird und für einen ABC-Transporter kodiert. Wir haben diesen Transporter Aat benannt (für acidic amino acid transporter). Aat wird hochreguliert, wenn P. putida auf sauren Aminosäuren oder deren Amiden wächst. Das AatPMQJ-System ist ein Transporter für polare Aminosäuren und gehört zur Gruppe 3 der Familie der periplasmatischen Ligandenbindungsproteine (SBP_Bac_3). Der Aat-Transporter besteht aus vier Untereinheiten, dem Nucleotid bindenden Protein AatP, zwei die Membran durchspannenden Permease-Einheiten (AatM und Aat Q) sowie dem periplasmatischen Bindeprotein AatJ. Rekombinant exprimiertes und gereinigtes AatJ zeigte hohe Affinität zu Glu und Asp (Kd = 0.4 μM bzw. 1.3 μM), während Gln und Asn und andere Dicarboxylate mit viel geringerer Affinität gebunden werden. Eine (auf Grundlge des Glutamin bindenden Proteins GlnH von E. coli) durch Homologie¬modelling erzeugte AatJ-Struktur legte nahe, dass sich sein Ligandenbindungszentrum von dem anderer Bindeproteine unterscheidet, wobei mehrere Argininreste maßgeblich sind. Die modellierte Struktur wurde experimentell bestätigt, indem diverse Aminosäurereste, die nach dem Modell an Wechselwirkungen mit dem Liganden beteiligt sein sollten, durch gerichtete Mutagenese verändert wurden. Eine Suche in Sequenzdatenbanken zeigte, dass AatJ zusammen mit mindestens 15 weiteren Proteinen eine neue Subfamilie der periplasmatischen Bindeproteine bildet. Die Beteiligung des Zweikomponentensystems AauRS an der Regulation der Transkription von aat und ansB (dem für PGA kodierenden Gen) wurde mit Hilfe des EMSA (electrophoretic mobility shift assay) nachge¬wiesen. Dabei zeigte sich, dass gereinigtes AauR an beide Promotoren bindet. Die genaue Bindungsstelle wurde durch DNAaseI-„Footprints“ identifiziert. Sie besteht in einem hoch konservierten Inverted Repeat aus je 6 bp, die durch 4 bp getrennt sind (GTTCGGNNNNCCGAAC). Bei einer in-silico Suche im P. putida KT2440-Genom wurden zusätzliche Gene entdeckt, die ebenfalls das AauR-Bindemotiv in ihrer Promoterregion enthalten. Zu den Produkten dieser Gene gehören ein H+/Glu-Symporter (GltP), das Enzym Phosphoenolpyruvat-Synthase (PpsA), ein ABC-Transporter für verzweigtkettige Aminosäuren (Bra) and das Disulfidaustauschprotein DsbC. Auf der Grundlage dieser Ergebnisse war es erstmals möglich, am Beispiel P. putida KT2440 konkrete Vorstellungen zur Aufnahme und Metabolisierung der sauren Aminosäuren und ihrer Regulation durch das AauRS Zweikom¬ponen¬tensystem zu entwickeln.
... In contrast, isomerization of the s 54 -RNAP-promoter closed complex requires interaction with the specific enhancer-bound, ATP-dependent activator (reviewed by Merrick, 1993;Shingler, 1996). Contact between the activator and the s 54 -RNAP-promoter complex is achieved by DNA looping, facilitated either by the integration host factor (IHF) protein or by intrinsic DNA topology (Pérez-Martin et al., 1994;Carmona et al., 1997). Control, imposed on the DNA melting step, requires ATP hydrolysis and involves the promoter 212/211 element (Guo et al., 1999(Guo et al., , 2000 bound inside the RNAP channel (Polyakov et al., 1995;Zhang et al., 1999;Severinov, 2000;Foster et al., 2001). ...
The Escherichia coli rpoH gene is transcribed from four known and differently regulated promoters: P1, P3, P4 and P5. This study demonstrates that the conserved consensus sequence of the sigma54 promoter in the regulatory region of the rpoH gene, described previously, is a functional promoter, P6. The evidence for this conclusion is: (i) the specific binding of the sigma54-RNAP holoenzyme to P6, (ii) the location of the transcription start site at the predicted position (C, 30 nt upstream of ATG) and (iii) the dependence of transcription on sigma54 and on an ATP-dependent activator. Nitrogen starvation, heat shock, ethanol and CCCP treatment did not activate transcription from P6 under the conditions examined. Two activators of sigma54 promoters, PspF and NtrC, were tested but neither of them acted specifically. Therefore, PspFDeltaHTH, a derivative of PspF, devoid of DNA binding capability but retaining its ATPase activity, was used for transcription in vitro, taking advantage of the relaxed specificity of ATP-dependent activators acting in solution. In experiments in vivo overexpression of PspFDeltaHTH from a plasmid was employed. Thus, the sigma54-dependent transcription capability of the P6 promoter was demonstrated both in vivo and in vitro, although the specific conditions inducing initiation of the transcription remain to be elucidated. The results clearly indicate that the closed sigma54-RNAP-promoter initiation complex was formed in vitro and in vivo and needed only an ATP-dependent activator to start transcription.
... A simple interpretation of the data described above is that the greater sensitivity of the low-affinity promoters to loss of ppGpp and DksA is due to poor occupancy by the σ 54 - RNAP available. However, optimal localization of the activator via IHF-mediated DNA bending is particularly important for transcriptional initiation from low-affinity σ 54 promoters that are rarely occupied by σ 54 -RNAP (Hoover et al., 1990; Claverie-Martin and Magasanik, 1992; Santero et al., 1992; Carmona et al., 1997). As IHF levels are partially under the control of ppGpp and show an abrupt increase at the exponential-to-stationary phase transition (Aviv et al., 1994; Ditto et al., 1994; Valls et al., 2002), we considered that the influence of ppGpp and DksA on IHF levels might contribute to the differences in dependence of the hybrid promoters on ppGpp/DksA in vivo. ...
The RNA polymerase-binding protein DksA is a cofactor required for guanosine tetraphosphate (ppGpp)-responsive control of transcription from sigma70 promoters. Here we present evidence: (i) that both DksA and ppGpp are required for in vivo sigma54 transcription even though they do not have any major direct effects on sigma54 transcription in reconstituted in vitro transcription and sigma-factor competition assays, (ii) that previously defined mutations rendering the housekeeping sigma70 less effective at competing with sigma54 for limiting amounts of core RNA polymerase similarly suppress the requirement for DksA and ppGpp in vivo and (iii) that the extent to which ppGpp and DksA affect transcription from sigma54 promoters in vivo reflects the innate affinity of the promoters for sigma54-RNA polymerase holoenzyme in vitro. Based on these findings, we propose a passive model for ppGpp/DksA regulation of sigma54-dependent transcription that depends on the potent negative effects of these regulatory molecules on transcription from powerful stringently regulated sigma70 promoters.
... Interaction between enhancer bound activator and promoter bound Es 54 is often face-of-the-helix dependent, suggesting the formation of a DNA loop between the enhancer and promoter. This DNA loop, in the case of the glnAp2 promoter, is due to intrinsic DNA bending (Carmona et al., 1997) and has been visualized directly by electron microscopy (Su et al., 1990). Up to date, there is no reported binding site between the enhancer and promoter of glnAp2 for DNA bending proteins. ...
... In earlier studies, Manuel Carmona and his colleague showed that the initiation of transcription on supercoiled or linear glnAp2, which has high affinity for Es 54 , does not require a single intrinsic or induced bend in the DNA. However, the transcription initiation at a promoter with low affinity for Es 54 , such as glnHp2, requires such an intrinsic or IHF induced DNA bend as well as DNA supercoiling (Carmona et al., 1997). These observations support the view that IHF stabilizes a DNA conformational change (DNA bending) that was needed for the productive interaction between Es 54 and the activator, and such contacts would increase the amount of open complexes by stimulating isomerization from closed to open complexes (Hoover et al., 1990). ...
A binding site for the Escherichia coli nucleoid binding protein FIS (factor for inversion stimulation) was identified upstream of a sigma54-dependent promoter, glnAp2. The binding and bending center of FIS is positioned at -55 with respect to the transcription start site (+1). Binding of FIS at this site activates the transcription of glnAp2 both in vivo and in vitro. Furthermore, we substituted the FIS-mediated DNA bending with other protein (cAMP receptor protein or integration host factor)-mediated DNA bending, without changing the position of the bending center. In vitro transcription assays indicated that all DNA bends centered at -55 activate transcriptional initiation of glnAp2, especially when linear templates were used.
