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A Kolb

Publications and source records attributed to A Kolb.

At least 37 records · Page 2Linked to original sources
PubMed2000

Positioning of region 4 of the Escherichia coli RNA polymerase sigma(70) subunit by a transcription activator.

A DNA cleavage reagent, specifically tethered to residue 581 of the Escherichia coli RNA polymerase sigma(70) subunit, has been used to investigate the location of sigma(70) region 4 in different complexes at the galp(1) promoter and the effect of the cyclic AMP receptor protein. The positions of DNA cleavage by the reagent are not affected by the cyclic AMP receptor protein. We conclude that transcription activation at the galp(1) promoter by the cyclic AMP receptor protein does not involve major conformation changes in or repositioning of sigma(70) region 4.

Bacterial Proteins↗
PubMed1999

Positioning of sigma(S), the stationary phase sigma factor, in Escherichia coli RNA polymerase-promoter open complexes.

The sigma(S) subunit of RNA polymerase is the master regulator of the general stress response in Escherichia coli and is required for promoter recognition of many stationary phase genes. We have analysed open complexes of Esigma(S) RNA polymerase, using sigma(S) derivatives carrying single cysteine residues at nine different positions to which the reagent FeBABE has been tethered. All holoenzymes but one formed transcriptionally active open complexes at three different promoters (osmY, galP1 and lacUV5). The chemical nuclease FeBABE can cleave DNA in proximity to the chelate. The overall cutting pattern of Esigma(S) open complexes does not depend on the nature of the promoter and is similar to that obtained with Esigma(70), but extends towards the downstream part of the promoter. The strongest cleavages are observed with FeBABE positioned on cysteines in regions 2.2 to 3.1. In contrast to sigma(70), region 2.1 of sigma(S) appears to be far from DNA. Region 4.2 of sigma(S) appears less accessible than its counterpart in sigma(70) and FeBABE positioned in the turn of the helix-turn-helix (HTH) motif in region 4.2 reacts only weakly with the -35 promoter element. This provides a structural basis for the minor role of the -35 sequence in sigma(S)-dependent promoter recognition.

Amino Acid Substitution↗
PubMed1999

[Oxygen saturation of retinal vessels. Studies for measuring with polarized light].

UNLABELLED: The accuracy of the spectrometric measurement of the oxygen saturation in retinal vessels is limited by its signal-to-noise ratio. The aim of this study was to investigate the possibility of enhancement of the reflection signal by the use of polarized light. MATERIALS AND METHODS: The Jena ophthalmospectrometer was equipped with two polarizing filters: one in the illumination and the other in front of the detector. Reflection spectra of erythrocytes streaming through a cuvette in the focus of an artificial eye were recorded. The influence of the polarization on the reflection spectra was investigated by rotating the polarizer in front of the detector. Furthermore, the degree of polarization of the light reflected from retinal vessels in vivo was determined. RESULTS: The degree of polarization of the light reflected from the erythrocytes was 0.6-0.8, whereas the polarization of light reflected by a standard white reflectance target was virtually zero. CONCLUSION: Polarized light can be used for the reduction of error in retinal vessel oximetry.

Blood Flow Velocity↗
PubMed1999

Multiple control of flagellum biosynthesis in Escherichia coli: role of H-NS protein and the cyclic AMP-catabolite activator protein complex in transcription of the flhDC master operon.

Little is known about the molecular mechanism by which histone-like nucleoid-structuring (H-NS) protein and cyclic AMP-catabolite activator protein (CAP) complex control bacterial motility. In the present paper, we show that crp and hns mutants are nonmotile due to a complete lack of flagellin accumulation. This results from a reduced expression in vivo of fliA and fliC, which encode the specific flagellar sigma factor and flagellin, respectively. Overexpression of the flhDC master operon restored, at least in part, motility in crp and hns mutant strains, suggesting that this operon is the main target for both regulators. Binding of H-NS and CAP to the regulatory region of the master operon was demonstrated by gel retardation experiments, and their DNA binding sites were identified by DNase I footprinting assays. In vitro transcription experiments showed that CAP activates flhDC expression while H-NS represses it. In agreement with this observation, the activity of a transcriptional fusion carrying the flhDC promoter was decreased in the crp strain and increased in the hns mutant. In contrast, the activity of a transcriptional fusion encompassing the entire flhDC regulatory region extending to the ATG translational start codon was strongly reduced in both hns and crp mutants. These results suggest that the region downstream of the +1 transcriptional start site plays a crucial role in the positive control by H-NS of flagellum biosynthesis in vivo. Finally, the lack of complementation of the nonmotile phenotype in a crp mutant by activation-deficient CAP mutated proteins and characterization of cfs, a mutation resulting in a CAP-independent motility behavior, demonstrate that CAP activates flhDC transcription by binding to its promoter and interacting with RNA polymerase.

Bacterial Proteins↗
PubMed1998

DNA bending and expression of the divergent nagE-B operons.

Repression of the divergent nagE - B operons requires NagC binding to two operators which overlap the nagE and nagB promoters, resulting in formation of a DNA loop. Binding of the cAMP/CAP activator to its site, adjacent to the nagE operator, stabilizes the DNA loop in vitro. The DNA of the nagE-B intergenic region is intrinsically bent, with the bend centred on the CAP site. We show that displacement of the CAP site by 6 bp results in complete derepression of the two operons. This derepression is observed even in the absence of cAMP/CAP binding and despite the fact that the two NagC operators are still in phase, demonstrating that the inherently bent structure of the DNA loop is important for repression. Since no interaction between NagC and CAP has been detected, we propose that the role of CAP in the repression loop is architectural, stabilizing the intrinsic bend. The cAMP/CAP complex is necessary for activation of the nagE-B promoters. In this case protein-protein contacts between CAP and RNA polymerase are necessary for full activation, but at least a part of the activation is likely due to an effect of CAP binding altering DNA structure.

Acetylglucosamine↗
PubMed1998

Molecular analysis of the regulation of csiD, a carbon starvation-inducible gene in Escherichia coli that is exclusively dependent on sigma s and requires activation by cAMP-CRP.

The general stress-induced sigma subunit sigma s of Escherichia coli RNA polymerase is closely related to the vegetative sigma factor sigma 70. In view of their very similar promoter specificity in vitro, it is unclear how sigma factor selectivity in the expression of sigma s-dependent genes is generated in vivo. The csiD gene is such a strongly sigma s-dependent gene. In contrast to sigma s, which is induced in response to many different stresses, csiD, whose expression is driven from a single promoter, is induced by carbon starvation only. To our knowledge, the csiD promoter is the first characterized promoter which is not only exclusively dependent on sigma s-containing RNA polymerase (E sigma s), but also requires an activator, cAMP-CRP. In addition, leucine-responsive regulatory protein (Lrp) acts as a positive modulator of csiD expression. Also in vitro, E sigma s is more efficient than E sigma 70 in csiD promoter binding, open complex formation and run-off transcription, which might be due to the poor match of the csiD -35 region to the sigma 70 consensus and to transcription by E sigma s being less dependent on contacts in this region. By DNase I protection experiments, a cAMP-CRP binding site centered at -68.5 nucleotides upstream of the csiD transcriptional start site was identified. While cAMP-CRP stimulates E sigma 70 binding, it does not promote open complex formation by E sigma 70, but does so in conjunction with E sigma s. With linear templates, cAMP-CRP significantly stimulates E sigma s-mediated in vitro transcription, whereas transcription by E sigma 70 is negligible and hardly stimulated by cAMP-CRP. These findings may reflect different or less stringent positional requirements for an activator site for E sigma s than for E sigma 70, and indicate that cAMP-CRP contributes to sigma factor selectivity at the csiD promoter. In vitro transcription experiments with super-coiled templates, however, revealed significant cAMP-CRP-stimulated transcription also by E sigma 70. Yet, under these conditions, H-NS was found to restore E sigma s specificity by strongly interfering with cAMP-CRP/E sigma 70-dependent transcription. Lrp strongly and cooperatively binds to multiple sites located between positions -14 and -102 (in a way that suggests DNA wrapping around multiple Lrp molecules) and moderately stimulates in vitro transcription, especially with E sigma s. In summary, we conclude that the csiD promoter has an intrinsic preference for E sigma s, but that also protein factors such as cAMP-CRP, Lrp and probably H-NS as well as DNA conformation contribute to its strong E sigma s selectivity. Furthermore, this strong E sigma s preference in combination with a requirement for high concentrations of the essential activator cAMP-CRP ensures csiD expression under conditions of carbon starvation, but not other stress conditions.

Bacterial Proteins↗
PubMed1998

CRP interacts with promoter-bound sigma54 RNA polymerase and blocks transcriptional activation of the dctA promoter.

The cAMP receptor protein (CRP) is an activator of sigma70-dependent transcription. Analysis of the sigma54-dependent dctA promoter reveals a novel negative regulatory function for CRP. CRP can bind to two distant sites of the dctA promoter, sites which overlap the upstream activator sequences for the DctD activator. CRP interacts with Esigma54 bound at the dctA promoter via DNA loop formation. When the CRP-binding sites are deleted, CRP still interacts in a cAMP-dependent manner with the stable Esigma54 closed complex via protein-protein contacts. CRP is able to repress activation of the dctA promoter, even in the absence of specific CRP-binding sites. CRP affects both the final level and the kinetics of activation. The establishment of the repression and its release by the NtrC activator proceed via slow processes. The kinetics suggest that CRP favours a new form of closed complex which interconverts slowly with the classical closed intermediate. Only the latter is capable of interacting with an activator to form an open promoter complex. Thus, Esigma54 promoters are responsive to CRP, a protein unrelated to sigma54 activators, and the repression exerted is the direct result of an interaction between Esigma54 and the CRP-cAMP complex.

Bacterial Proteins↗
PubMed1998

The bacteriophage T4 AsiA protein: a molecular switch for sigma 70-dependent promoters.

The AsiA protein, encoded by bacteriophage T4, inhibits Esigma70-dependent transcription at bacterial and early-phage promoters. We demonstrate that the inhibitory action of AsiA involves interference with the recognition of the -35 consensus promoter sequence by host RNA polymerase. In vitro experiments were performed with a C-terminally labelled sigma factor that is competent for functional holoenzyme reconstitution. By protease and hydroxyl radical protein footprinting, we show that AsiA binds region 4.2 of sigma70, which recognizes the -35 sequence. Direct interference with the recognition of the promoter at this locus is supported by two parallel experiments. The stationary-phase sigma factor containing holoenzyme, which can initiate transcription at promoters devoid of a -35 region, is insensitive to AsiA inhibition. The recognition of a galP1 promoter by Esigma70 is not affected by the presence of AsiA. Therefore, we conclude that AsiA inhibits transcription from Escherichia coli and T4 early promoters by counteracting the recognition of region 4.2 of sigma70 with the -35 hexamer.

Amino Acid Sequence↗
PubMed1997

The interaction between the AsiA protein of bacteriophage T4 and the sigma70 subunit of Escherichia coli RNA polymerase.

The AsiA protein of bacteriophage T4 binds to the sigma70 subunit of Escherichia coli RNA polymerase and plays a dual regulatory role during T4 development: (i) inhibition of host and phage early transcription, and (ii) coactivation of phage middle-mode transcription, which also requires the T4 DNA binding transcriptional activator, MotA. We report that the interaction between AsiA and sigma70 occurs with a 1:1 stoichiometry. When preincubated with RNA polymerase, AsiA is a potent inhibitor of open complex formation at the lac UV5 promoter, whereas it does not perturb preformed open or intermediate promoter complexes. DNase I footprinting and electrophoretic mobility shift analyses of RNA polymerase-DNA complexes formed at the T4 early promoter P15.0 show that AsiA blocks the initial RNA polymerase binding step that leads to the formation of specific closed promoter complexes. A contrasting result is obtained on the T4 middle promoter PrIIB2, where AsiA stimulates the formation of both closed complexes and open complexes. Therefore, we propose that AsiA modulates initial DNA binding by the RNA polymerase, switching promoter usage at the level of closed complex formation.

Bacterial Proteins↗
PubMed1997

Nucleoprotein complex formation by the enhancer binding protein nifA.

The nitrogen fixation protein NifA is a member of the protein family activating transcription by the alternative eubacterial sigmaN (sigma54) RNA polymerase holoenzyme. Binding sites for NifA, upstream activator sequences (UASs), are remotely located. Interaction between holoenzyme bound in a closed promoter complex and NiFA is facilitated by bending of the intervening DNA by integration host factor (IHF). We have examined NifA contact with the Klebsiella pneumoniae nifH promoter UAS in the presence and absence of holoenzyme and IHF. Footprints with UV light were made on 5-BrdU-substituted DNA and DNase I and laser UV footprints on conventional DNA templates. Results establish that the consensus thymidine residues of the UAS motif 5'-TGT are in close proximity to NifA. Reactivity suggests that each UAS thymidine is not structurally equivalent. Titration of NifA binding to the UAS in the presence or absence of the closed promoter complex indicates that the interaction of NifA with the UAS is not strongly co-operative with holoenzyme or IHF, a result supportive of an activation mechanism not reliant upon simple recruitment of factors to the promoter. Laser footprints demonstrated that holoenzyme suppressed reactivity of promoter consensus -14, -15 and -16 T residues, indicating close contact. Binding of holoenzyme resulted in a specific increase in 5-BrdU reactivity at -9 within the holoenzyme binding site, likely reflecting DNA distortion. Enhanced -9 reactivity required sigmaNN-terminal sequences that are necessary for activation. Since T-9 is melted in open complexes the closed complex appears poised for melting. Open promoter complex formation was accompanied by a distinct change in laser footprint signal at -11, consistent with the view that nucleation of strand separation occurs within or close to the -12 promoter element.

Azotobacter vinelandii↗
PubMed1997

DNA flexibility of the UP element is a major determinant for transcriptional activation at the Escherichia coli acetate promoter.

The specific interaction of the upstream element-containing promoter of the Escherichia coli acetate operon with either the RNA polymerase holoenzyme or its alpha subunit has been analyzed by the base removal method. Our results indicate that: (i) direct and specific base contacts can be detected in the acetate promoter-alpha subunit complex; (ii) base elimination in the upstream element of the acetate promoter enhances the binding of RNA polymerase. A similar effect is observed when studying the interactions between RNA polymerase and the rrnB ribosomal operon P1 promoter.

Acetates↗
PubMed1996

Orientation of functional activating regions in the Escherichia coli CRP protein during transcription activation at class II promoters.

At class II CRP-dependent promoters the DNA site for CRP overlaps the DNA site for RNA polymerase, covering the -35 region. Transcription activation at class II CRP- dependent promoters requires a contact between an activating region in the upstream subunit of the bound CRP dimer and a contact site in the C-terminal domain of the alpha-subunit of RNA polymerase. Transcription activation is suppressed by amino acid substitutions in the activating region, but activation can be restored by second site substitutions at K52 or E96. These substitutions identify two separate regions on the surface of CRP that appear to be able to interact with RNA polymerase specifically at class II promoters. Using the method of 'oriented heterodimers' we show that these alternative activating regions are functional in the downstream subunit of the bound CRP dimer.

Bacterial Proteins↗
PubMed1996

Factors influencing the timing of peripheral blood stem cell collection (PBSC).

High-dose conditioning regimens followed by autologous peripheral blood stem cell rescue are frequently used for the treatment of solid tumors and hematological malignancies. In 24 patients up to four peripheral stem cell collections (PBSC) were performed after priming with various chemotherapies and G-CSF (300 micrograms s.c. per day). In 16 patients (group A) more than 2 x 10(6) CD 34 positive cells per kg bodyweight could be collected; fewer were harvested in the remaining eight patients (group B). The amount of collected CD 34 positive cells correlated with the median number of these cells in the peripheral blood at the start of PBSC. The two groups differed both in recovery time after priming-induced cytopenia (4 vs 6 days from nadir) and in the number of WBC (21 x 10(6) mL-1 vs 6.1 x 10(6) mL-1) and platelets (133 x 10(6) mL-1 vs 58 x 10(6) mL-1) reached at first day of PBSC. No difference between the two groups was seen according to age, duration of disease or disease status. However, the intensity of prior treatment was significantly greater in group B than in group A. These observations indicate that the toxicity of previous chemotherapy is the most important factor for the mobilization of sufficient CD 34 positive cells into the peripheral blood.

Adolescent↗
PubMed1995

Nag repressor-operator interactions: protein-DNA contacts cover more than two turns of the DNA helix.

The NagC repressor binds to two sites in the intergenic nagE-B region overlapping the divergently expressed nagE and nagB promoters. In addition the NagC repressor binds to two sites upstream of the manXYZ operon. Although basically palindromic, there is little sequence consensus between the four operators. To identify the DNA sequence important for NagC recognition, we have taken advantage of the fact that repression of the nagE and nagB genes requires the formation of a loop of DNA between molecules of the repressor bound to the nagE and nagB operators. The nagE operator was systematically mutagenised and the effect of the mutations measured on the level of expression from a nagB-lacZ fusion. These experiments showed that the most important positions for recognition are the two A.T base-pairs at positions-5 and -6 from the centre of symmetry. These are the only absolutely conserved bases in the four operators. Certain changes of residues at position -3 and -4 have fairly strong effects while changes at -7 to -10 have only minor effects. However the presence of a G or C base at positions + 11 or -11 produces a NagC binding site with considerably higher affinity than the wide-type nagE operator both in vitro and in vivo, a "super-operator". The presence of a super-operator considerably increased the stability of the binary looped NagC-DNA complex in vitro. However in the presence of cAMP/CAP, NagC showed the same apparent binding affinity to wild-type and super-operators indicating that one role of cAMP/CAP in the repression complex is to reduce the need for high affinity sites. These super-operators allow a higher level of repression of the nagE promoter compared to the nagB, presumably due to the existence of linear complexes of NagC bound to BoxE.

Bacterial Proteins↗
PubMed1995

Selectivity of the Escherichia coli RNA polymerase E sigma 38 for overlapping promoters and ability to support CRP activation.

A series of gal promoter mutants has been used to compare the in vitro selectivities of the two forms of Escherichia coli RNA polymerase, E sigma 38 and E sigma 70. In the absence of the CRP-cAMP complex, E sigma 38 shows a strong preference for the ga/P1 promoter, whereas E sigma 70 preferentially initiates transcription from the ga/P2 promoter. E sigma 38 selectivity is not affected by the nature and position of the upstream sequences or by the phasing between synthetic upstream curved sequences and the -10 regions. In fact, all effects of mutations in the extended -10 region can be accounted for without evoking strong new sequence preferences for E sigma 38. Finally, both E sigma 38 and E sigma 70 initiate transcription from the ga/P1 promoter in the presence of CRP-cAMP complex and support direct cAMP-CRP activation at several CRP-dependent promoters.

Bacterial Proteins↗
PubMed1994

CRP fixes the rotational orientation of covalently closed DNA molecules.

Five minicircles of 284 bp were constructed with a reporter sequence located approximately opposite a CRP binding site. The spacing between the center of the CRP site and this sequence is varied within 1.2 helical turns. The reactivity of the reference sequence to DNAse I was determined on the minicircles and on the corresponding fragments, in both the absence and presence of CRP. A rigorous mathematical analysis of the data shows that in the absence of CRP no preferred rotational orientation of the DNA is observed. In contrast, binding of CRP fixes, in a phase-dependent manner, the rotational orientation of the reporter sequence in the minicircles. This result illustrates the transmission at a distance along the DNA molecule of a structural modification. Such effects modulate the extent of synergy between activators and polymerases during the initiation of transcription.

Binding Sites↗
PubMed1994

Transcription activation by the Escherichia coli cyclic AMP receptor protein. Receptors bound in tandem at promoters can interact synergistically.

Starting with a semi-synthetic Escherichia coli promoter with a binding site for the cyclic AMP receptor protein (CRP) centred between base-pairs 41 and 42 upstream from the transcription start site, a second upstream CRP-binding site, centred between base-pairs 90 and 91, was introduced. CRP binding to this second upstream site results in a several-fold greater stimulation of CRP-dependent transcription initiation, compared to activation at the starting promoter with just one CRP-binding site. Activation of transcription by the upstream CRP molecule is blocked by the HL159 substitution, suggesting that the upstream-bound CRP makes a direct contact with RNA polymerase. Footprinting experiments suggest that RNA polymerase contacts the promoter DNA between the two CRP-binding sites, most likely due to interactions involving the C-terminal part of the alpha subunit. Synergy between tandem bound CRP molecules in transcription activation requires that the two CRP-binding sites be separated by around 40 or 50 base-pairs, but is not found at intermediate spacings. An experiment in which the upstream CRP-binding site is replaced by a site for the related transcription factor, FNR, shows that heterologous synergistic interactions between FNR and CRP are possible.

Bacterial Proteins↗