1,721,059 research outputs found
Interaction of the main cold shock protein CS7.4 (CspA) of Escherichia coli with the promoter region of hns
Full text linkEscherichia coli protein CS7.4 (CspA), homologous to the class of eukaryotic Y-box DNA-binding proteins, is a cold shock transcriptional activator of at least two genes, hns and gyrA. It was demonstrated that all or nearly all the elements necessary for the stimulation of hns transcription by CS7.4 protein are located in the proximal 110 bp DNA fragment of this gene with no additional elements being present in a longer fragment (660 bp) extending further upstream from the hns promoter. Protein CS7.4 bound strongly to the 110 bp segment of the hns promoter in crude extracts of cold shocked cells, but the purified protein displayed a weak interaction with the same DNA fragment. Purified CS7.4 protein also caused increased or decreased accessibility to DNase I at different sites of the 110 bp fragment of hns but the majority of these effects was seen only in the presence of RNA polymerase. Since gel shift experiments showed that protein CS7.4 stimulated the binding of RNA polymerase to the promoter of hns and since it is known that there are similarities between CS7.4 and ssDNA-binding proteins, we suggest that formation of the open complex by the RNA polymerase or protein-protein contacts between CS7.4 and the RNA polymerase are prerequisites for and/or the effects of the interaction of CS7.4 with its DNA target. The presence of a conserved CCAAT element in the hns promoter region, on the other hand, was found not to be stringently required for cold shock activation since expression of E coli of an hns-cat fusion containing the Proteus vulgaris hns promoter lacking a CCAAT box increased over four-fold after cold shock
How to cope with the quest for new antibiotics
No full textSince their introduction in therapy, antibiotics have played an essential role in human society, saving millions of lives, allowing safe surgery, organ transplants, cancer therapy. Antibiotics have also helped to elucidate several biological mechanisms and boosted the birth and growth of pharmaceutical companies, generating profits and royalties. The golden era of antibiotics and the scientific and economical drive of big pharma towards these molecules is long gone, but the need for effective antibiotics is increased as their pipelines dwindle and multi-resistant pathogenic strains spread. Here we outline some strategies that could help meet this emergency and list promising new targets
PROTEINS FROM THE PROKARYOTIC NUCLEOID.II. INHIBITION OF DNA TRANSCRIPTION BY NS1 AND NS2 (HU PROTEINS)
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Kinetic checkpoint at a late step in translation initiation
No full textThe translation initiation efficiency of a given mRNA is determined by its translation initiation region (TIR). mRNAs are selected into 30S initiation complexes according to the strengths of the secondary structure of the TIR, the pairing of the Shine-Dalgarno sequence with 16S rRNA, and the interaction between initiator tRNA and the start codon. Here, we show that the conversion of the 30S initiation complex into the translating 70S ribosome constitutes another important mRNA control checkpoint. Kinetic analysis reveals that 50S subunit joining and dissociation of IF3 are strongly influenced by the nature of the codon used for initiation and the structural elements of the TIR. Coupling between the TIR and the rate of 70S initiation complex formation involves IF3- and IF1-induced rearrangements of the 30S subunit, providing a mechanism by which the ribosome senses the TIR and determines the efficiency of translational initiation of a particular mRNA
Environmental control of the in vivo oligomerization of nucleoid protein H-NS
No full textThe nucleoid-associated transcriptional repressor H-NS forms both dimers and tetramers in vivo. Two types of two-hybrid systems, one capable of detecting protein dimerization and the other protein tetramerization, have been used to determine whether environmental changes could affect the oligomerization capacity of this protein in the cell. Increasing the temperature from 37°C to 48°C and changing the pH between 4.0 and 9.0 did not influence either dimerization or tetramerization, whereas lowering the temperature below 25°C and increasing osmolarity were found to reduce the formation of H-NS tetramers, which are the active form of this protein, without affecting dimerization. These findings provide a rationale to explain the induction of H-NS expression during cold-shock, suggest a mechanism contributing to derepressing osmotic-shock genes transcriptionally regulated by H-NS and indicate that changes of the oligomerization properties of H-NS do not play a role in the H-NS and temperature-dependent control of virulence gene expression
Preferential translation of cold-shock mRNAs during cold adaptation
No full textUpon temperature downshift below the lower threshold of balanced growth (∼20°C), the Escherichia coli translational apparatus undergoes modifications allowing the selective translation of the transcripts of cold shock-induced genes, while bulk protein synthesis is drastically reduced. Here we were able to reproduce this translational bias in E. coli cell-free extracts prepared at various times during cold adaptation which were found to display different capacities to translate different types of mRNAs as a function of temperature. Several causes were found to contribute to the cold-shock translational bias: Cold-shock mRNAs contain cis-elements, making them intrinsically more prone to being translated in the cold, and they are selective targets for trans-acting factors present in increased amounts in the translational apparatus of cold-shocked cells. CspA was found to be among these trans-acting factors. In addition to inducing a higher level of CspA, cold shock was found to cause a strong (two-to threefold) stoichiometric imbalance of the ratio between initiation factors (IF1, IF2, IF3) and ribosomes without altering the stoichiometric ratio between the factors themselves. The most important sources of cold-shock translational bias is IF3, which strongly and selectively favors translation of cold-shock mRNAs in the cold. IF1 and the RNA chaperone CspA, which stimulate translation preferentially in the cold without mRNA selectivity, can also contribute to the translational bias. Finally, in contrast to a previous claim, translation of cold-shock cspA mRNA in the cold was found to be as sensitive as that of a non-cold-shock mRNA to both chloramphenicol and kanamycin inhibition
PROTEIN FROM THE PROKARYOTIC NUCLEOID. I.EFFECT OF NS1 AND NS2 (HU) PROTEINS ON THE THERMAL STABILITY OF DNA.
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The oligomeric structure of nucleoid protein H-NS is necessary for recognition of intrinsically curved DNA and for DNA bending.
No full textMassive presence of the Escherichia coli 'major cold-shock protein' CspA under non-stress conditions
Full text linkThe most characteristic event of cold-shock activation in Escherichia coli is believed to be the de novo synthesis of CspA. We demonstrate, however, that the cellular concentration of this protein is > or = 50 microM during early exponential growth at 37 degrees C; therefore, its designation as a major cold-shock protein is a misnomer. The cspA mRNA level decreases rapidly with increasing cell density, becoming virtually undetectable by mid-to-late exponential growth phase while the CspA level declines, although always remaining clearly detectable. A burst of cspA expression followed by a renewed decline ensues upon dilution of stationary phase cultures with fresh medium. The extent of cold-shock induction of cspA varies as a function of the growth phase, being inversely proportional to the pre-existing level of CspA which suggests feedback autorepression by this protein. Both transcriptional and post-transcriptional controls regulate cspA expression under non-stress conditions; transcription of cspA mRNA is under the antagonistic control of DNA-binding proteins Fis and H-NS both in vivo and in vitro, while its decreased half-life with increasing cell density contributes to its rapid disappearance. The cspA mRNA instability is due to its 5' untranslated leader and is counteracted in vivo by the cold-shock DeaD box RNA helicase (CsdA)
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