Transcription Regulation in the hyperthermophilic crenarchaeon Thermoproteus tenax strain Kra1

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Archaeal transcription is generally regarded as simpler model of eukaryotic transcription (BALIGA et al., 2000). Relatively little is known about the interaction of gene-specific regulators and the basal transcription apparatus and about how transcriptional regulation in Archaea helps to confer fitness across a broad range of environments, including hostile ones. Multiple forms of the general transcription factors (GTFs), TBP and TFB, are commonly found in Archaea (LANGER et al., 1995). It has been suggested previously that multiple TBPs and TFBs might function similar to bacterial sigma-factors, which regulate transcription in response to environmental changes (PAYTUBI & WHITE, 2009). In the group of Crenarchaeota, only TFB1 from S. solfataricus and S. acidocaldarius (BELL & JACKSON, 2000) and recently, TFB3 from S. solfataricus (PAYTUBI & WHITE, 2009) have been studied biochemically. However, the role of TFB2 in Crenarchaeota is still unknown and no tfb knockout mutants are available for any of the crenarchaeal TFBs. The hyperthermophilic crenarchaeon T. tenax genome encodes one TBP and four TFBs. Bioinformatic analyses revealed that Ttx-TFB1 harbors a Zn-ribbon motif, B-finger and two cyclin domains similar to other TFB homologues in Archaea, however, Ttx-TFB2-4 possess significant modifications in secondary structure. In order to study the function of Ttx-GTFs, recombinant proteins were expressed in E. coli. EMSAs were performed using the fba-pfp promoter and revealed that Ttx-TFB1, as classical TFB, binds to DNA stable in a TBP-dependent manner whereas Ttx-TFB2 and Ttx-TFB3 shows stable DNA binding independent of TBP. To our knowledge, this is the first report of a TFB homologue binding DNA independent of TBP. Exo III footprinting analyses using the fba-pfp promoter (encoding fructose-1,6-bisphophate aldolase and PPi-dependent phosphofructokinase in T. tenax (SIEBERS et al., 2004)) illustrate that Ttx-TFB1 binds to the core promoter (from position -38 to -21), which is stabilized by Ttx-TBP. However, for Ttx-TFB2, no binding to the BRE/TATA box of the fba-pfp promoter region and no influence of Ttx-TBP is observed. Furthermore, Ttx-TFB1 and Ttx-TFB2 bind in addition to a sequence (from position -101 to -84) with high similarity to BRE/TATA box of the archaeal consensus promoter (SLUPSKA et al., 2001). Mutations of the fba-pfp promoter region affected the stability of the binding of Ttx-TFB1/Ttx-TBP complex to DNA suggesting that the complex binds unstable to the region -101/-84. The stable binding of Ttx-TFB2 to the region -101/-84 suggest that this region might function similar to an upstream activation sequence (UAS) for the fba-pfp promoter of T. tenax. Therefore, these results suggest that Ttx-TFB2 does not function as basal transcription factor (i.e. typical TFIIB), but rather as transcriptional activator; similar to the function of Sso-TFB3 (PAYTUBI & WHITE, 2009). Multiprotein bridging factor 1 (MBF1) is a transcriptional co-activator that bridges a sequence-specific activator and TBP in Eukaryotes. MBF1 is absent in Bacteria, but is well- conserved in Eukaryotes and Archaea and harbors a C-terminal Cro-like Helix Turn Helix (HTH) domain, which is the only highly conserved, classical HTH domain that is vertically inherited in all Eukaryotes and Archaea. Phylogenetic analyses revealed a common distribution of MBF1 in all Archaea with available genome sequence. The main structural difference between archaeal MBF1 (aMBF1) and eukaryotic MBF1 is the presence of a Zn-ribbon motif in aMBF1. The function of co-activators from Archaea has been not studied so far and the biological role of aMBF1 as multiprotein bridging factor has not been documented. In yeast the MBF1 mediated activation of histidine synthesis by the bZIP-like activator GCN4 has been well studied (TAKEMARU et al., 1998). To study the function and therefore the evolutionary conservation of MBF1 and its single domains, complementation studies in yeast (mbf1Δ) as well as domain swap experiments between aMBF1 and yMBF1 were performed. In contrast to previous reports for eukaryal MBF1 (i.e. Arabidopsis thaliana, insect and human), the two archaeal MBF1 orthologs, TMBF1 from the hyperthermophile T. tenax and MMBF1 from the mesophile M. mazei were not functional for complementation of a yeast mutant lacking MBF1 (mbf1Δ). From 12 chimeric proteins representing different combinations of the N-terminal, core domain, and the C-terminal extension from yeast and aMBF1, only the chimeric MBF1 comprising the yeast N-terminal and the core domain fused to the archaeal C-terminal part was able to restore full wild-type activity of MBF1. The yMBF1 mutant lacking the C-terminal part was also able to restore MBF1 activity, suggesting that this part is not important for MBF1 function. The absence of bZIP-like proteins in the archaeal domain, the presence of a Zn-ribbon in the divergent N-terminal domain of aMBF1 and the complementation experiments using Archaea- yeast chimeric proteins presented here suggests no obvious conservation in biological function of MBF1 within Archaea and Eukaryotes. It is tempting to speculate that aMBF1 might act as a single regulator, which directly interacts with DNA for transcriptional regulation via its Zn-ribbon motif. To our knowledge, this is the first experimental study to gain insight about the physiological function of MBF1 in Archaea.
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Wissenschaftliche Abschlussarbeiten » Dissertation
Fakultät / Institut:
Fakultät für Chemie » Biofilm Center
Dewey Dezimal-Klassifikation:
500 Naturwissenschaften und Mathematik » 540 Chemie
transcription, MBF1, TFB, archaea
Prof. Dr. rer. nat. Siebers, Bettina [Betreuer(in), Doktorvater]
Prof. Dr. Ehrenhofer-Murray, Ann [Gutachter(in), Rezensent(in)]
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