Promoter structure, promoter recognition, and transcription activation in prokaryotes.
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DNA-Directed RNA Polymerases↗
Biomedical subjects
S Busby
Publications and source records attributed to S Busby.
Interactions between the cyclic AMP receptor protein and the alpha subunit of RNA polymerase at the Escherichia coli galactose operon P1 promoter.
DNAase I footprinting has been used to study open complexes between Escherichia coli RNA polymerase and the galactose operon P1 promoter, both in the absence and the presence of CRP (the cyclic AMP receptor protein, a transcription activator). From the effects of deletion of the C-terminal part of the RNA polymerase alpha subunit, we deduce that alpha binds at the upstream end of both the binary RNA polymerase-galP1 and ternary RNA polymerase-CRP-galP1 complexes. Disruption of the alpha-upstream contact suppresses open complex formation at galP1 at lower temperatures. In ternary RNA polymerase-CRP-galP1 complexes, alpha appears to make direct contact with Activating Region 1 in CRP. DNAase I footprinting has been used to detect and quantify interactions between purified alpha and CRP bound at galP1.
Base Sequence↗
The functional subunit of a dimeric transcription activator protein depends on promoter architecture.
In Class I CAP-dependent promoters, the DNA site for CAP is located upstream of the DNA site for RNA polymerase. In Class II CAP-dependent promoters, the DNA site for CAP overlaps the DNA site for RNA polymerase, replacing the -35 site. We have used an 'oriented heterodimers' approach to identify the functional subunit of CAP at two Class I promoters having different distances between the DNA sites for CAP and RNA polymerase [CC(-61.5) and CC(-72.5)] and at one Class II promoter [CC(-41.5)]. Our results indicate that transcription activation at Class I promoters, irrespective of the distance between the DNA sites for CAP and RNA polymerase, requires the activating region of the promoter-proximal subunit of CAP. In striking contrast, our results indicate that transcription activation at Class II promoters requires the activating region of the promoter-distal subunit of CAP.
Bacterial Proteins↗
Noise levels in New Zealand junior classrooms: their impact on hearing and teaching.
AIMS: To observe the acoustic conditions present in classrooms populated by the 5 to 7 year old age group, and investigate the level of understanding amongst primary school teachers of classroom acoustics and otitis media with effusion. METHODS: Fifty one primary schools in the Wellington region were visited. Acoustic conditions were observed in 106 classrooms. 149 questionnaires were given to the teachers. Sound level readings were taken during normal school activities. Teacher's voice and background noise measurements were recorded. The signal to noise (S/N) ratios obtained were analysed in conjunction with the acoustic conditions observed. RESULTS: The median S/N ration was +6dB. This is a poor S/N ratio. Analysis revealed that poor S/N ratios were associated with a high background noise level and multifunctional teaching activities. CONCLUSIONS: The acoustic conditions in the majority of classrooms studied were unacceptable.
Acoustics↗
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↗
Interactions between the Escherichia coli MelR transcription activator protein and operator sequences at the melAB promoter.
The Escherichia coli MelR protein binds to two sites at the melAB promoter and activates transcription in response to melibiose. The binding of purified MelR to the melAB promoter and to a number of derivatives carrying point mutations has been studied. A melAB promoter fragment that is very weakly activated by the melR gene product has been made by creating multiple symmetrical changes in both MelR-binding sites. A library of random substitutions in MelR was screened to find mutants that were able to activate this mutant promoter. One of these mutants contained a substitution in a proposed helix-turn-helix region that most likely relaxes the sequence-specificity of MelR binding. The other mutants contain substitutions throughout the central part of MelR and appear to alter the conformation of MelR, such that activity is triggered at lower melibiose concentrations and by arabinose.
Base Sequence↗
Location and orientation of an activating region in the Escherichia coli transcription factor, FNR.
We have characterized a number of mutations in fnr that interfere with FNR-dependent transcription activation at two promoters where the FNR-binding site is centred around 41 1/2 bp upstream from the transcription start site. The substituted residues in all but one of these FNR mutants are clustered around a presumed surface-exposed beta-turn containing G85 which, we suggest, forms an activating region that contacts RNA polymerase at these promoters. Using the 'oriented heterodimers' method described elsewhere, we show that this activating region on the promoter-proximal subunit of the FNR dimer is sufficient to activate transcription initiation. In contrast, this region is not essential for activation of a third FNR-dependent promoter where the FNR-binding site is centred at 61 1/2 bp upstream from the transcription start site. However, a substitution at S73 interferes with FNR-dependent activation at both this promoter and promoters in which the FNR site is located at 41 1/2 bp from the transcript start, suggesting that FNR may contain a second activating region.
Amino Acid Sequence↗
A seven-gene operon essential for formate-dependent nitrite reduction to ammonia by enteric bacteria.
The DNA sequence of the regulatory region and the structural gene, nrfA, for cytochrome c552 of Escherichia coli K-12 have been reported. We have now established that nrfA is the first gene in a seven-gene operon, designated the nrf operon, at least five of which are essential for formate-dependent nitrite reduction to ammonia. This operon terminates just upstream of the previously sequenced gltP gene encoding a sodium-independent, glutamate and aspartate transporter. Expression of lac fused to nrfA, nrfE or nrfG is regulated by oxygen repression, FNR-dependent anaerobic induction, nitrite induction and nitrate repression during anaerobic growth, exactly as previously reported for the nrfA promoter. In contrast, expression of the gltP-lac fusion was FNR-independent. The open reading frame immediately downstream of nrfA encodes NrfB, a hydrophilic, penta-haem cytochrome c with an M(r) of 20,714. The structure of the N-terminal region is typical of a signal peptide for a periplasmic protein: cleavage at the putative signal peptide cleavage site, Ala-26, would result in a periplasmic cytochrome with a molecular mass of 18 kDa. The NrfC polypeptide, M(r) 24,567, contains 16 cysteine residues arranged in four clusters typical of the CooF super-family of non-haem iron-sulphur proteins. The NrfD sequence predicts a 318-residue hydrophobic protein with a distribution of acidic and basic amino acids which suggests that NrfD is an integral transmembrane protein with loops in both the periplasm and the cytoplasm. Proteins most similar to NrfD include the PsrC subunit of polysulphide reductase from Wolinella, but, as seven of the 10 most similar proteins are NADH-ubiquinone oxidoreductases, we propose that NrfD participates in the transfer of electrons from the quinone pool into the terminal components of the Nrf pathway. NrfE, M(r) 60,851, is predicted to be another hydrophobic, integral membrane protein homologous to the CdI1 protein of Rhodobacter capsulatus, which has been implicated in the assembly of periplasmic c-type cytochromes. The sequence of the 127 residue NrfF polypeptide, M(r) 14,522, is strikingly similar to the CcI2 protein of R. capsulatus, especially in the putative haem-binding motif, RCPQCQNQN.(ABSTRACT TRUNCATED AT 400 WORDS)
Amino Acid Sequence↗
Assays for transcription factor activity.
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Binding Sites↗
The Escherichia coli cysG promoter belongs to the 'extended -10' class of bacterial promoters.
The Escherichia coli cysG promoter has been subcloned and shown to function constitutively in a range of different growth conditions. Point mutations identify the -10 hexamer and an important 5'-TGN-3' motif immediately upstream. The effects of different deletions suggest that specific sequences in the -35 region are not essential for the activity of this promoter in vivo. This conclusion was confirmed by in vitro run-off transcription assays. The DNAase I footprint of RNA polymerase at the cysG promoter reveals extended protection upstream of the transcript start, and studies with potassium permanganate as a probe suggest that the upstream region is distorted in open complexes. Taken together, the results show that the cysG promoter belongs to the 'extended -10' class of promoters, and the base sequence is similar to that of the P1 promoter of the E. coli galactose operon, another promoter in this class. In vivo, messenger initiated at the cysG promoter appears to be processed by cleavage at a site 41 bases downstream from the transcript start point.
Base Sequence↗
Investigation of the Pseudomonas aeruginosa ampR gene and its role at the chromosomal ampC beta-lactamase promoter.
The complete sequence of the Pseudomonas aeruginosa beta-lactamase regulator gene ampR has been determined. The amino acid sequence encoded by this gene is compared with other members of the LysR family of transcription factors. The P. aeruginosa ampC promoter was subcloned and gel mobility shift assays were used to demonstrate specific binding of AmpR to the ampC promoter region.
Amino Acid Sequence↗
An evaluation of the potential to use tumor-associated antigens as targets for antitumor T cell therapy using transgenic mice expressing a retroviral tumor antigen in normal lymphoid tissues.
A major obstacle to the development of T cell therapy for the treatment of human tumors has been the difficulty generating T cells specifically reactive with the tumor. Most of the characterized human tumor antigens have been classified as tumor associated, because of demonstrable expression at low levels in some normal cells, and thus have not been extensively studied as potential targets of a therapeutic immune response. However, the quantitative difference in expression of such antigens between the tumor and normal cells might permit the generation of antigen-specific T cells capable of selective antitumor and not autoimmune activity. To address this issue, transgenic (TG) mice were generated that expressed low levels of Friend murine leukemia virus (FMuLV) envelope protein in lymphoid cells under the control of an immunoglobulin promoter. This protein is expressed at high levels by a Friend virus-induced erythroleukemia of C57BL/6 (B6) origin, FBL, and has been shown to serve as an efficient tumor-specific rejection antigen in B6 mice. The env-TG mice were tolerant to envelope, as reflected by the failure to detect an envelope-specific response after in vivo priming and in vitro stimulation with preparations of FMuLV envelope. However, adoptively transferred envelope-specific T cells from immunized non-TG B6 mice mediated complete eradication of FBL tumor cells in TG mice, and did not induce detectable autoimmune damage to TG lymphoid tissues. The transferred immune cells were not permanently inactivated in the TG mice, since donor T cells responded to envelope after removal from the TG mice. The lack of autoimmune injury did not reflect inadequate expression of envelope by TG lymphocytes for recognition by T cells, since TG lymphocytes functioned effectively in vitro as stimulators for envelope-specific T cells. The results suggest that this and analogous strains of TG mice may prove useful for elucidating principles for the generation and therapeutic use of tumor-reactive T cells specific for tumor-associated antigens.
Animals↗
Identification of the functional subunit of a dimeric transcription activator protein by use of oriented heterodimers.
We have constructed heterodimers consisting of two subunits: one CAP subunit that has a nonfunctional activating region but wild-type DNA binding specificity, and one CAP subunit that has a functional activating region but non-wild-type DNA binding specificity. We have oriented the heterodimers on lac promoter DNA by use of promoter derivatives that have DNA sites for CAP consisting of one wild-type half site and one non-wild-type half site, and we have analyzed the abilities of the oriented heterodimers to activate transcription. Our results indicate that transcription. Our results indicate that transcription activation requires the activating region of only one subunit of CAP: the promoter-proximal subunit. The oriented heterodimers method of this report should be generalizable to other dimeric transcription activator proteins.
Bacterial Proteins↗
Location of close contacts between Escherichia coli RNA polymerase and guanine residues at promoters either with or without consensus -35 region sequences.
Methylation-interference assays have been used to identify guanine residues that make important contacts with RNA polymerase during open-complex formation at two related Escherichia coli promoters. Methylation of lower-strand G-31 at a gal consensus promoter completely prevents complex formation, while modification of upper-strand G-33 has no detectable effect. At galP1, which lacks a consensus -35 region, modification of lower-strand G-33 and upper-strand G-14 reduces, but does not prevent, complex formation. G-33 is the only guanine residue in the -35 region of galP1 where modification interferes with open-complex formation. Since this guanine residue is not protected in open complexes, we conclude that its modification causes alteration of, or interference with, a transient contact during the transcription initiation pathway.
Base Composition↗
Interactions between the Escherichia coli cyclic AMP receptor protein and RNA polymerase at class II promoters.
The effects of a number of mutations in crp have been measured at different cyclic AMP receptor protein (CRP)-dependent Class II promoters, where the CRP-binding site is centred around 41 1/2 base pairs upstream from the transcription start point. The amino acid substitutions HL159 and TA158 result in reduced CRP-dependent activation, but the reduction varies from one Class II promoter to another. Deletions in the C-terminus of the RNA polymerase alpha subunit suppress the effects of HL159 and TA158. The role of the C-terminus of alpha at these promoters is assessed. Other changes at E58, K52 and E96 affect CRP activity specifically at Class II promoters and their role is discussed.
Amino Acid Sequence↗
Regulation and sequence of the structural gene for cytochrome c552 from Escherichia coli: not a hexahaem but a 50 kDa tetrahaem nitrite reductase.
The structural gene, nrfA, for cytochrome c552, which is the terminal reductase of the formate-dependent pathway for nitrite reduction to ammonia, has been located at co-ordinate 4366 on the physical map of the Escherichia coli chromosome. The DNA sequence of nrfA encodes a tetrahaem c-type cytochrome with a predicted M(r) for the unprocessed product of 53,788. Cleavage of the putative signal peptide at Ala-26 would result in a mature, periplasmic cytochrome of M(r) 50,580 rather than a larger hexahaem cytochrome, as has been widely reported previously. A cytochrome of this size was detected by staining SDS-polyacrylamide gels for covalently bound haem. This cytochrome was partially purified by anion exchange chromatography and confirmed to be cytochrome c552 by difference spectroscopy. Similar cytochromes were detected in five other E. coli strains including strain ST 249, which was used previously to purify and characterize the protein. A plasmid with an in-phase deletion within nrfA directed the synthesis of a truncated haemoprotein of the predicted mass. In-phase translational fusions to lacZ were used to locate the nrfA translation start, and the transcription start site was found by S1 mapping. Expression from the FNR-dependent nrfA promoter was almost totally repressed during aerobic growth, partially induced during anaerobic growth in the absence of nitrite or in the presence of nitrate, but fully induced only during anaerobic growth in the presence of nitrite. No nitrate repression was detected in a narL mutant, but nitrite induction was unaffected, indicating that the nitrite-sensing mechanism is independent of the NarL protein. Expression from the nrfA promoter was subject to glucose repression but regulation was independent of the CRP-cAMP complex.
Aerobiosis↗
Transcriptional regulation by cAMP and its receptor protein.
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Allosteric Regulation↗
Immunoaffinity chromatography in biorecovery: an application of recombinant DNA technology to generic adsorption processes.
The constant region of human kappa light chain (Ck) was linked to Escherichia coli beta-galactosidase, using standard molecular cloning techniques. The binding of Ck-beta-galactosidase fusions to a number of different murine monoclonal antibodies, specific for Ck, was improved by the insertion of spacers between Ck and beta-galactosidase: a cleavable linker was then introduced. Over-expressed Ck-beta-galactosidase fusion protein was purified using monoclonal antibodies immobilised on Sepharose 4B. Elution conditions were found that maintained beta-galactosidase activity so purified enzyme could be released on breaking the cleavable linker. A number of practical problems associated with maintaining stable fusion proteins and immunoaffinity column performance were identified.
Adsorption↗