Biochemical and functional characterization of the HP1043 orphan response regulator of the human pathogen Helicobacter pylori

Pelliciari, Simone (2018) Biochemical and functional characterization of the HP1043 orphan response regulator of the human pathogen Helicobacter pylori, [Dissertation thesis], Alma Mater Studiorum Università di Bologna. Dottorato di ricerca in Biologia cellulare e molecolare, 30 Ciclo. DOI 10.6092/unibo/amsdottorato/8561.
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In Helicobacter pylori, the hp1043 gene is one of the transcriptional regulator essential for cell viability. As such, this gene could not be deleted, supporting the hypothesis that HP1043 could be involved in the regulation of crucial cellular processes. The impossibility of generating a knock-out mutant for hp1043 gene, or even modulate the amount of HP1043 protein in the cell, has hampered the detailed characterization of its regulatory function. Using Chromatin Immunoprecipitation-sequencing (ChIP-seq), we were able to identify genome-wide at least 37 new HP1043 binding sites. Moreover, in vitro DNase I protection assays (footprints) enabled mapping of the HP1043 binding sites on a subset of the new targets, revealing the presence of a conserved nucleotide sequence motif consisting of a direct TTTAAG repeat. Furthermore, hydroxyl-radical probing allowed to further refine the positions of HP1043 binding, suggesting that the proposed direct repeats consensus motif is recognized by HP1043, likely through a major groove read-out mechanism. Intriguingly, a significant fraction of newly identified binding sites overlaps promoter regions of genes involved in translation. Accordingly, arrest of protein translation determined induction of almost all HP1043 target genes. These observations prompted us to propose HP1043 as key regulator in H. pylori, likely involved in sensing and in coordinating the response to environmental stress inducing an arrest of protein synthesis. DNase I and hydroxyl-radical footprinting experiments aimed to elucidate the role of each base of the consensus motif in the protein-DNA binding were performed. They showed a fundamental role of both hemisite, with major effect on the AAG out of TTTAAG for the first sequence and TTAA hexamer core for the second. Experiments aimed to elucidate the functional role of the protein were carried out by in vitro transcription assays, to evaluate the effect of HP1043 promoter binding on the activity on the RNA polymerase.

Tipologia del documento
Tesi di dottorato
Pelliciari, Simone
Dottorato di ricerca
Settore disciplinare
Settore concorsuale
Parole chiave
Helicobacter pylori, HP1043, transcriptional regulation
Data di discussione
18 Aprile 2018

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