Browsing by Subject "Genregulation"

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Functional characterization of the COOH-terminal kinase activity of the TBP-associated factor TAF1
(2006) Maile, Tobias; Sauer, Frank
Activation of eukaryotic transcription involves an orchestrated interplay between transcription factors and the general RNA polymerase II (Pol II) transcription machinery (GTM), which consists of Pol II and general transcription factors (GTFs). The GTF TFIID consists of the TATA-box binding protein (TBP) and several TBP-associated factors (TAFs). The binding of TFIID to promoters can nucleate transcription. TAF1 is the largest subunit of TFIID and plays a central role within the nucleating function of TFIID in transcription. TAF1 mediates the binding of TFIID to promoters and interacts with enhancer-bound transcription factors and several GTFs. Additionally, TAF1 contains four enzymatic activities that are essential for viability of eukaryotes and mediate posttranslational modification of GTFs and histones. TAF1 is a bipartite protein kinase and contains an NH2-terminal kinase domain (NTK) and a COOH-terminal kinase domain (CTK). A previous study demonstrated that the CTK phosphorylates serine-residue 33 in histone H2B (H2BS33). However, the role of TAF1-mediated phosphorylation in transcription regulation remained unknown. In this study, the functional importance of H2BS33 phosphorylation (H2BS33P) by TAF1 was investigated by using a combination of biochemical and in vivo assays. In vitro kinase assays uncovered the two essential kinase motifs in TAF1CTK, the ATP-binding motif and the serine/threonine-specific catalytic motif, and indicate that the TAF1 CTK has intrinsic kinase-activity. Western blot analysis using an antibody to H2BS33P revealed that H2BS33 is phosphorylated in Drosophila. RNA-interference (RNAi) assays, designed to attenuate TAF1 expression (TAF1RNAi), revealed that TAF1 is a major kinase for H2BS33 in Drosophila Schneider cells. Flow-cytometry analysis of TAF1RNAi cells indicated that loss of TAF1 expression results in cell cycle arrest in G2/M-phase. Screening the transcription of cell cycle genes in TAF1RNAi cells by using reverse-transcriptase-PCR demonstrated that the transcription of the cell cycle gene string (stg) is reduced in the absence of TAF1. Chromatin immunoprecipitation assays (XChIP) indicate that H2BS33P is detectable at the transcriptionally active stg promoter but not at the silent stg promoter in TAF1RNAi cells. These results demonstrate that phosphorylation of H2BS33 is involved in stg transcription. XChIP-assays using chromatin prepared from Drosophila embryos, which express a mutant TAF1 lacking the CTK, revealed that CTK-mediated phosphorylation of H2BS33 plays an essential role in the activation of transcription of the Drosophila segmentation gene giant. In vitro kinase assays demonstrate that Bdf1 and Bdf2, the yeast homologues of the TAF1CTK, phosphorylate histones suggesting that the kinase activity of the TAF1CTK is phylogenetically conserved. The results of this work demonstrate that TAF1CTK is a major histone kinase of H2BS33 and that TAF1-mediated phosphorylation of H2BS33 plays an essential role in the transcription events during cell cycle progression and development.
Molekulargenetische Untersuchungen zur Expression des Typ III Effektors NleA 4795 von Shiga Toxin-produzierenden Escherichia coli
(2010) Schwidder, Maike; Schmidt, Herbert
Shiga toxin-producing E. coli (STEC) are the causative agents of foodborne infections in many countries and can lead to severe diseases like hemorrhagic colitis or the life-threatening hemolytic uremic syndrome. The bacteria colonize the human intestine where they normally cause the formation of characteristic ?attaching and effacing?-lesions. Essential for this effect is a pathogenicity island, termed as ?locus of enterocyte effacement? (LEE), that encodes the components of a type III secretion system and several effector proteins, which are translocated directly into the host cells by the TTSS machinery. In addition to the LEE-encoded effectors a large number of effector proteins have been identified which are encoded outside of the pathogenicity island. Among these is the ?non-LEE encoded effector A? (NleA), which is encoded on cryptic or inducible prophages and is widely distributed among pathogenic E. coli strains. In the present study, the expression and regulation of the nleA-variant nleA4795 of E. coli O84:H4 strain 4795/97 was investigated, which is located on the Shiga toxin-converting bacteriophage BP-4795. Therefore, different environmental conditions as well as certain regulatorproteins were tested on their influence on nleA4795-expression using a luciferase-reportersystem and the quantitative real-time PCR. Among the analyzed environmental factors, certain concentrations of NaCl and KCl were identified to activate nleA4795-expression, indicating an osmotic-based influence. The suggested induction of nleA4795 in preconditioned medium due to quorum sensing could not be confirmed, since none of the so far known autoinducers showed a positive influence on the expression. The increased expression of nleA4795 could be associated with a reduced amount of nutrients in subsequent investigations and therefore demonstrated a relation between nleA4795-expression and bacterial stress-response-systems. Furthermore, a possible correlation of nleA4795-expression with the induction of phage BP 4795 and Shiga toxin-expression was analyzed. Different from the expression of Shiga toxin, induction-experiments with norfloxacin showed no activation, but a strong repression of nleA4795-expression. Analysis of the regulatory level demonstrated that the expression of nleA4795 depends on the three LEE-encoded regulators Ler, GrlA und GrlR as well as on the Pch-regulators, which are encoded outside of the LEE. The non-LEE encoded regulator EtrA showed no influence on the expression of nleA4795. In addition, the regulator proteins Ler, GrlA and PchA were tested for direct binding to the nleA4795-promoterregion. Regulators GrlA and PchA showed no specific binding and were therefore classified as indirect regulators of nleA4795-expression. In contrast, regulator Ler showed a specific binding to different areas of the nleA4795-promoter region and thereby confirmed the integration of nleA4795 in the Ler-mediated circuit of LEE-regulation.