RESUMO
The interaction of RNA polymerase and its initiation factors is central to the process of transcription initiation. To dissect the role of this interface, we undertook the identification of the contact sites between RNA polymerase and sigma(70), the Escherichia coli initiation factor. We identified nine mutationally verified interaction sites between sigma(70) and specific domains of RNA polymerase and provide evidence that sigma(70) and RNA polymerase interact in at least a two-step process. We propose that a cycle of changes in the interface of sigma(70) with core RNA polymerase is associated with progression through the process of transcription initiation.
Assuntos
Proteínas de Ligação a DNA/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Fragmentos de Peptídeos/metabolismo , Fator sigma/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/genética , Genes Reporter , Immunoblotting , Modelos Moleculares , Fragmentos de Peptídeos/genética , Mutação Puntual , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fator sigma/química , Fator sigma/genética , Transcrição GênicaRESUMO
PURPOSE: To better understand the molecular program of neuronal cell death induced by axotomy, the authors attempted to identify retinal genes differentially expressed by optic nerve crush. METHODS: Total RNA isolated from rat retinas at 1 and 4 days after intraorbital optic nerve crush was used in a modification of the differential display technique. After several rounds of screening, a single reproducibly upregulated band was reamplified and cloned, and differential expression was confirmed by Northern analysis. RESULTS: Sequencing of the differentially expressed band revealed identity to the ferroxidase ceruloplasmin. Reverse transcription-polymerase chain reaction demonstrated high levels of ceruloplasmin expression in retina and liver, but minimal or no expression in brain, lung, spleen, kidney, or thymus of adult rats. The retina mRNA transcript was the same size as that of the liver, as measured by Northern blotting. In situ hybridization identified ceruloplasmin expression in the inner nuclear and ganglion cell layers of the retina, which increased after optic nerve crush. Immunoblotting confirmed expression of the same size protein product in the retina and the liver, and ceruloplasmin could be identified in the retina by immunofluorescence, which increased after optic nerve crush. CONCLUSIONS: Ceruloplasmin was expressed in the retina, and was induced by optic nerve crush. The possible role of ceruloplasmin in inhibiting reaction oxygen species in the retina after injury is discussed.
Assuntos
Ceruloplasmina/biossíntese , Compressão Nervosa , Nervo Óptico/fisiologia , Retina/metabolismo , Animais , Axotomia , Northern Blotting , Ceruloplasmina/genética , Primers do DNA/química , Técnica Indireta de Fluorescência para Anticorpo , Regulação da Expressão Gênica , Hibridização In Situ , Fígado/metabolismo , Nervo Óptico/cirurgia , Reação em Cadeia da Polimerase , RNA/isolamento & purificação , RNA Mensageiro/biossíntese , Ratos , Células Ganglionares da Retina/metabolismoRESUMO
PURPOSE: Retinal ganglion cells die by apoptosis after axotomy, and this process may reflect altered expression of cell-death genes. Several of these genes, including bcl-2, bcl-x, and bax, share homology at the amino acid level in the BH1 and BH2 domains, through which they also interact. To understand their role in the neuronal response to axotomy, the authors studied their expression in the adult rat retina and after optic nerve crush. METHODS: An initial survey was conducted with reverse transcription-polymerase chain reaction (RT-PCR), using oligonucleotides against identified members of this family and against the conserved BH1 and BH2 domains. Retinal bcl-xl expression at the messenger RNA (mRNA) and protein level was studied by RT-PCR, Northern blotting, RNase protection analysis, in situ hybridization, Western blotting, and immunofluorescence staining. The effect of retinal ganglion cell axotomy on the steady-state level of bcl-x mRNA was investigated. RESULTS: RT-PCR results indicated that rat retinal cells predominantly express the long form of bcl-x. Both clonal analysis and quantitative measurements using RNase protection assays demonstrated that bcl-xL message was at least 16 times more abundant than that of bcl-2. In situ hybridization and indirect immunofluorescence demonstrated that nearly all neuronal cells of the retina express bcl-x. Northern and RNase protection analyses showed a moderate decrease in bcl-xL message shortly after optic nerve crush. CONCLUSIONS: These findings suggest that the antideath gene bcl-xL is the predominant member of the bcl-2 family in the adult retina, and that its level decreases after optic nerve crush. Changes in bcl-xL expression may correlate with increased retinal ganglion cell apoptosis after axotomy.