RESUMO
Expression of the Escherichia coli StpA protein was investigated and a functional comparison undertaken with the structurally analogous nucleoid protein H-NS. Analysis of stpA and hns expression indicated that although stpA transcript levels are much lower than those of hns, the two gene products are capable of both negative autogenous control and cross-regulation. Examination of cellular proteins in stpA, hns, or stpA-hns backgrounds revealed that StpA can repress and activate a subset of H-NS-regulated genes. Mechanistic parallels in regulation of gene expression are indicated by the ability of both proteins to inhibit transcription from promoters containing curved DNA sequences, and to form nucleoprotein structures that constrain DNA supercoils. Despite their functional similarities, each molecule is capable of independent activities. Thus, H-NS regulates a class of genes that are unaffected by StpA in vivo, whereas StpA has much stronger RNA chaperone activity in vitro. We therefore propose that in addition to its role as a molecular back-up of H-NS, StpA's superior effect on RNA may be exploited under some specific cellular conditions to promote differential gene expression.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Chaperonas Moleculares , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Sequência de Bases , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/genética , Modelos Genéticos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Splicing de RNA , RNA Bacteriano/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transcrição GênicaAssuntos
Bactérias/genética , Proteínas de Bactérias/genética , Íntrons/genética , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Splicing de RNA , Archaea/genética , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Modelos Genéticos , Filogenia , Células ProcarióticasRESUMO
We previously showed that the in vitro transcription of a negatively supercoiled plasmid containing the murine IgA switch region caused the formation of fewer supercoiled conformers of the plasmid due to the presence of a stable RNA.DNA hybrid. Here, we demonstrate that the RNA.DNA hybrid is approximately 140 nucleotides, and it forms regardless of the initial topological state of the transcription template. Transcription of the switch region in a relaxed closed circular plasmid generates positively supercoiled plasmid conformers that revert to their original relaxed state when treated with RNase H. Conformers that have incorporated a stable RNA transcript are also observed when nicked circular and linear plasmids containing the IgA switch sequences are transcribed with 32P-labeled nucleotide triphosphates. Once formed, the RNA.DNA hybrid is stable to both thermal and superhelical stress, tolerating temperatures in excess of 95 degrees C and restraining approximately 12 positive supercoils in the plasmid.
Assuntos
Rearranjo Gênico , Imunoglobulina A/genética , Região de Troca de Imunoglobulinas/genética , Ácidos Nucleicos Heteroduplexes , Transcrição Gênica , Animais , Sequência de Bases , RNA Polimerases Dirigidas por DNA/metabolismo , Modelos Genéticos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Desnaturação de Ácido Nucleico , Hibridização de Ácido Nucleico , Nucleotídeos/análise , Proteínas ViraisRESUMO
A deletion DNA rearrangement is associated with immunoglobulin class switching from IgM to IgG, IgA or IgE This recombination occurs in immunoglobulin switch regions, which are complex, highly repetitive regions of DNA. As switch regions become transcriptionally active just before switch recombination, analysis of the behaviour of these sequences during transcription could elucidate the mechanism of switch recombination. Here, we report that transcription of a supercoiled plasmid containing the murine IgA switch region (S alpha) leads to a loss of superhelical turns. The resulting series of less supercoiled plasmids is stabilized by RNA-DNA hybrids formed by the nascent RNA transcripts, which remain base-paired with their DNA templates.
Assuntos
DNA/genética , Imunoglobulina A/genética , Imunoglobulina G/genética , Imunoglobulina M/genética , Região de Troca de Imunoglobulinas/genética , RNA/genética , Transcrição Gênica , Composição de Bases , Sequência de Bases , Deleção Cromossômica , Humanos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , Plasmídeos , Recombinação Genética , Mapeamento por RestriçãoRESUMO
We report the detection of low-density lipoprotein (LDL) receptors by the technique of receptor blotting in fibroblasts from a patient with homozygous familial hypercholesterolemia (FHC) previously classified as "receptor negative." Solubilized receptors were electrophoresed, transferred to nitrocellulose paper, treated with LDL followed by radiolabeled antibody to LDL, and visualized by autoradiography. GM 2000 FHC fibroblasts revealed LDL receptors with an apparent molecular weight of approximately 140,000, the same as in normal cells. LDL receptor activity by blotting in GM 2000 cells was greatly diminished in comparison with normal cells, but was calcium dependent. Receptor activity was also detectable by conventional monolayer binding and degradation assays. Thus, GM 2000 cells have profoundly diminished LDL receptor activity, but retain the genetic capacity to make LDL receptor material of normal molecular weight that is capable of binding LDL. Previous studies have demonstrated the presence of trace amounts of immunoreactive LDL receptor protein in fibroblasts from some receptor-negative FHC homozygotes. Our work extends these studies by demonstrating the ability of this material to bind LDL.
