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Oxygen sensitivity of the nifLA promoter of Klebsiella pneumoniae.   总被引:4,自引:3,他引:1       下载免费PDF全文
Q T Kong  Q L Wu  Z F Ma    S C Shen 《Journal of bacteriology》1986,166(1):353-356
Oxygen sensitivity of the nifLA promoter of Klebsiella pneumoniae has been demonstrated. Studies on the oxygen regulation of nifB-lacZ and nifH-lacZ fusions in the presence of the nifLA operon, which contains either an intact or a deleted nifL gene, indicate that possibly both the nifL promoter and the nifL product are responsible for nif repression by oxygen.  相似文献   

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The Klebsiella pneumoniae nifU promoter is positively controlled by the NifA protein and requires a form of RNA polymerase holoenzyme containing the rpoN encoded sigma factor, sigma 54. Occupancy of the K. pneumoniae nifU promoter by NifA was examined using in vivo dimethyl sulphate footprinting. Three binding sites for NifA (Upstream Activator Sequences, UASs 1, 2 and 3) located at -125, -116 and -72 were identified which conform to the UAS consensus sequence TGT-N10-ACA. An additional NifA binding site was identified at position -90. The UASs located at -125 (UAS1) and -116 (UAS2) overlap and do not appear to bind NifA as independent sites. They may represent a NifA binding site interacting with two NifA dimers. UAS3 is located at -72, and abuts a binding site for integration host factor (IHF) and is not normally highly occupied by NifA. In the absence of IHF UAS3 showed increased occupancy by NifA. Mutational and footprinting analysis of the three UASs indicates (1) IHF and NifA can compete for binding and that this competition influences the level of expression from the nifU promoter (2) that UAS2 is a principle sequence of the UAS 1,2 region required for activation and (3) that none of the NifA binding sites interacts with NifA independently. In vivo KMnO4 footprinting demonstrated that NifA catalyses open complex formation at the nifU promoter. IHF was required for maximal expression from the nifU and nifH promoters in Escherichia coli, and for the establishment of a Nif+ phenotype in E. coli from the nif plasmid pRD1.  相似文献   

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F Govantes  E Andújar    E Santero 《The EMBO journal》1998,17(8):2368-2377
The nifLA operon of Klebsiella pneumoniae encodes the sensor-activator pair involved in the regulation of other nif genes. Balanced synthesis of both proteins, which is required for correct regulation, is achieved by coupling translation of nifA to that of nifL. The mechanism of translational coupling at the nifLA operon was analysed using a specialized ribosome system, and the effect of substituting the natural Shine-Dalgarno of nifL or nifA for specialized Shine-Dalgarno sequences was determined. Our results indicate that translational coupling occurs in this operon by a reinitiation mechanism. Additionally, reinitiation at the nifA can happen even in the absence of good Shine-Dalgarno recognition by the reinitiating ribosome, although its efficiency is lower. The effect of a putative translational enhancer sequence (downstream box) on translational coupling efficiency was also determined. Mutations that reduce the homology of the putative downstream box to the consensus had only a minor effect on nifA translation by wild-type ribosomes. However, they had a significant effect on nifA translation by specialized ribosomes, suggesting that recognition of the downstream box may compensate inefficient ribosomal interactions with the Shine-Dalgarno sequence.  相似文献   

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In the present study, we have analyzed an upstream regulatory element of the neural cell adhesion molecule (NCAM) promoter which is required for full promoter activity. It contains an ATTATTA motif that resembles the core recognition sequence of homeodomain (HD) proteins of the Antennapedia (Antp) and related types. Electrophoretic mobility shift (EMSA) and DNase I footprinting analyses revealed that the Drosophila HDs coded by the Antp and the zerknüllt (zen) genes bind this site in vitro. In contrast, the engrailed (en) protein did not produce a detectable footprint. The functional relevance of the ATTATTA motif was demonstrated by showing that a two-nucleotide exchange curtailed stimulation of an heterologous promoter. An oligonucleotide known to be recognized with high affinity by Antp-like HDs efficiently competed for endogenous factor binding. These results suggest that the NCAM gene may be a target for HD proteins.  相似文献   

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The effect of catabolite activator protein, CAP, on the thermal stability of DNA was examined. Site specific binding was studied with a 62 bp DNA restriction fragment containing the primary CAP site of the E. coli lactose (lac) promoter. A 144 bp DNA containing the lac promoter region and a 234 bp DNA from the pBR322 plasmid provided other DNA sites. Thermal denaturation of protein-DNA complexes was carried out in a low ionic strength solvent with 40% dimethyl sulfoxide, DMSO. In this solvent free DNA denatured below the denaturation temperature of CAP. The temperature stability of CAP for site specific binding was monitored using an acrylamide gel electrophoresis assay. Results show that both specific and non-specific CAP binding stabilize duplex DNA. Site specific binding to the 62 bp DNA produced a 13.3 degrees C increase in the transition under conditions where non-specific binding stabilized this DNA by 2-3 degrees C.  相似文献   

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