Information theory was used to build a promoter model that accounts for the −10, the −35 and the uncertainty of the gap between them on a common scale. Helical face assignment indicated that base −7, rather than −11, of the −10 may be flipping to initiate transcription. We found that the sequence conservation of σ70 binding sites is 6.5 ± 0.1 bits. Some promoters lack a −35 region, but have a 6.7 ± 0.2 bit extended −10, almost the same information as the bipartite promoter. These results and similarities between the contacts in the extended −10 binding and the −35 suggest that the flexible bipartite σ factor evolved from a simpler polymerase. Binding predicted by the bipartite model is enriched around 35 bases upstream of the translational start. This distance is the smallest 5′ mRNA leader necessary for ribosome binding, suggesting that selective pressure minimizes transcript length. The promoter model was combined with models of the transcription factors Fur and Lrp to locate new promoters, to quantify promoter strengths, and to predict activation and repression. Finally, the DNA-bending proteins Fis, H-NS and IHF frequently have sites within one DNA persistence length from the −35, so bending allows distal activators to reach the polymerase.
Parkinson disease and other ␣-synucleinopathies are characterized by the deposition of intraneuronal ␣-synuclein (␣Syn) inclusions. A significant fraction (about 15%) of ␣Syn in these pathological structures are truncated forms that have a much higher propensity than the full-length ␣Syn to form aggregates in vitro. However, little is known about the role of truncated ␣Syn species in pathogenesis or the means by which they are generated. Here, we have provided an in vitro mechanistic study demonstrating that truncated ␣Syns induce rapid aggregation of full-length protein at substoichiometric ratios. Co-overexpression of truncated ␣Syn with full-length protein increases cell vulnerability to oxidative stress in dopaminergic SH-SY5Y cells. These results suggest a precipitating role for truncated ␣Syn in the pathogenesis of diseases involving ␣Syn aggregation. In this regard, the A53T mutation found in some cases of familial Parkinson disease exacerbates the accumulation of insoluble ␣Syns that correlates with the onset of pathology in transgenic mice expressing human ␣Syn-A53T mutant. The caspase-like activity of the 20 S proteasome produces truncated fragments similar to those found in patients and animal models from degradation of unstructured ␣Syn. We propose a model in which incomplete degradation of ␣Syn, especially under overloaded proteasome capacity, produces highly amyloidogenic fragments that rapidly induce the aggregation of full-length protein. These aggregates in turn reduce proteasome activity, leading to further accumulation of fragmented and full-length ␣Syns, creating a vicious cycle of cytotoxicity. This model has parallels in other neurodegenerative diseases, such as Huntington disease, where coaggregation of poly(Q) fragments with full-length protein has been observed.
A computational search was carried out to identify additional targets for the Escherichia coli OxyR transcription factor. This approach predicted OxyR binding sites upstream of dsbG, encoding a periplasmic disulfide bond chaperone-isomerase; upstream of fhuF, encoding a protein required for iron uptake; and within yfdI. DNase I footprinting assays confirmed that oxidized OxyR bound to the predicted site centered 54 bp upstream of the dsbG gene and 238 bp upstream of a known OxyR binding site in the promoter region of the divergently transcribed ahpC gene. Although the new binding site was near dsbG, Northern blotting and primer extension assays showed that OxyR binding to the dsbG-proximal site led to the induction of a second ahpCF transcript, while OxyR binding to the ahpCF-proximal site leads to the induction of both dsbG and ahpC transcripts. Oxidized OxyR binding to the predicted site centered 40 bp upstream of the fhuF gene was confirmed by DNase I footprinting, but these assays further revealed a second higher-affinity site in the fhuF promoter. Interestingly, the two OxyR sites in the fhuF promoter overlapped with two regions bound by the Fur repressor. Expression analysis revealed that fhuF was repressed by hydrogen peroxide in an OxyR-dependent manner. Finally, DNase I footprinting experiments showed OxyR binding to the site predicted to be within the coding sequence of yfdI. These results demonstrate the versatile modes of regulation by OxyR and illustrate the need to learn more about the ensembles of binding sites and transcripts in the E. coli genome.
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