RESUMO
Prions are self-propagating protein aggregates that are characteristically transmissible. In mammals, the PrP protein can form a prion that causes the fatal transmissible spongiform encephalopathies. Prions have also been uncovered in fungi, where they act as heritable, protein-based genetic elements. We previously showed that the yeast prion protein Sup35 can access the prion conformation in Escherichia coli. Here, we demonstrate that E. coli can propagate the Sup35 prion under conditions that do not permit its de novo formation. Furthermore, we show that propagation requires the disaggregase activity of the ClpB chaperone. Prion propagation in yeast requires Hsp104 (a ClpB ortholog), and prior studies have come to conflicting conclusions about ClpB's ability to participate in this process. Our demonstration of ClpB-dependent prion propagation in E. coli suggests that the cytoplasmic milieu in general and a molecular machine in particular are poised to support protein-based heredity in the bacterial domain of life.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Fatores de Terminação de Peptídeos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Endopeptidase Clp , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Choque Térmico/genética , Immunoblotting , Microscopia de Fluorescência , Chaperonas Moleculares/genética , Mutação , Fatores de Terminação de Peptídeos/genética , Príons/genética , Príons/metabolismo , Estabilidade Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Dodecilsulfato de Sódio/químicaRESUMO
Prions are infectious, self-propagating protein aggregates that have been identified in evolutionarily divergent members of the eukaryotic domain of life. Nevertheless, it is not yet known whether prokaryotes can support the formation of prion aggregates. Here we demonstrate that the yeast prion protein Sup35 can access an infectious conformation in Escherichia coli cells and that formation of this material is greatly stimulated by the presence of a transplanted [PSI(+)] inducibility factor, a distinct prion that is required for Sup35 to undergo spontaneous conversion to the prion form in yeast. Our results establish that the bacterial cytoplasm can support the formation of infectious prion aggregates, providing a heterologous system in which to study prion biology.
Assuntos
Escherichia coli/metabolismo , Fatores de Terminação de Peptídeos/metabolismo , Príons/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Escherichia coli/genética , Fatores de Terminação de Peptídeos/genética , Príons/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , SolubilidadeRESUMO
The sigma-subunit of bacterial RNA polymerase (RNAP) is required for promoter-specific transcription initiation. This function depends on specific intersubunit interactions that occur when sigma associates with the RNAP core enzyme to form RNAP holoenzyme. Among these interactions, that between conserved region 4 of sigma and the flap domain of the RNAP beta-subunit (beta-flap) is critical for recognition of the major class of bacterial promoters. Here, we describe the isolation of amino acid substitutions in region 4 of Escherichia coli sigma(70) that have specific effects on the sigma(70) region 4/beta-flap interaction, either weakening or strengthening it. Using these sigma(70) mutants, we demonstrate that the sigma region 4/beta-flap interaction also can affect events occurring downstream of transcription initiation during early elongation. Specifically, our results provide support for a structure-based proposal that, when bound to the beta-flap, sigma region 4 presents a barrier to the extension of the nascent RNA as it emerges from the RNA exit channel. Our findings support the view that the transition from initiation to elongation involves a staged disruption of sigma-core interactions.
Assuntos
RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , RNA Bacteriano/biossíntese , Fator sigma/química , Fator sigma/metabolismo , Substituição de Aminoácidos , Sítios de Ligação/genética , RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Genes Bacterianos , Holoenzimas/química , Holoenzimas/genética , Holoenzimas/metabolismo , Modelos Biológicos , Modelos Moleculares , Complexos Multiproteicos , Regiões Promotoras Genéticas , RNA Bacteriano/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fator sigma/genética , Transcrição GênicaRESUMO
The sigma(70) subunit of RNA polymerase plays an essential role in transcription initiation. In addition, sigma(70) has a critical regulatory role during transcription elongation at the bacteriophage lambda late promoter, lambda P(R'). At this promoter, sigma(70) mediates a pause in early elongation through contact with a DNA sequence element in the initially transcribed region that resembles a promoter -10 element. Here we provide evidence that sigma(70) also mediates a pause in early elongation at the lac promoter (plac). Like that at lambda P(R'), the pause at plac is facilitated by a sequence element in the initially transcribed region that resembles a promoter -10 element. Using biophysical analysis, we demonstrate that the pause-inducing sequence element at plac stabilizes the interaction between sigma(70) and the remainder of the transcription elongation complex. Bioinformatic analysis suggests that promoter-proximal sigma(70)-dependent pauses may play a role in the regulation of many bacterial promoters.
