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Ss-LrpB, a novel transcriptional regulator from the hyperthermoacidophilic crenarchaeon Sulfolobus solfataricus P2.

Wednesday, 25 April, 2007 - 16:00
Campus: Brussels Humanities, Sciences & Engineering campus
Faculty: Science and Bio-engineering Sciences
D
2.01
Eveline Peeters
phd defence

Although Archaea are true prokaryotes, transcription in these organisms resembles the eukaryotic process very closely. On the other hand, many transcription regulators appear to be Bacteria-like. How do these regulators function? This question remains largely unanswered, since information on archaeal transcription regulation is scarce. Many characterized archaeal species are extremophiles, including the hyperthermoacidophile Sulfolobus solfataricus P2, which grows optimally at 80°C and pH 2-3. In this organism, we identified a transcription regulator belonging to the bacterial/archaeal Leucine-responsive Regulatory Protein family (Lrp), called Ss-LrpB. The aim of this thesis was to characterize this protein, unravel its function (DNA-binding mode, physiological role) and structure as a contribution to a better understanding of archaeal transcriptional regulation.

Ss-LrpB binds its own control region in vitro at three regularly spaced, 15 bp-long binding sites, which is an observation suggestive of autoregulation. Each binding site is bound by one Ss-LrpB dimer. The two outer binding sites are bound with a higher affinity than the weaker, middle binding site which is only bound at higher Ss-LrpB concentrations. This interaction is highly cooperative. Atomic Force Microscopy allowed us to visualize Ss-LrpB-DNA complexes with a different stoichiometry and confirmed exhaustive DNA deformations upon protein binding, and even DNA wrapping when all three binding sites are occupied. The three binding sites appear to have a well conserved sequence. The deduced palindromic consensus sequence was the starting point of an extensive analysis (saturation mutagenesis) of in vitro binding to mutated consensus binding sites. This resulted in a detailed knowledge of the DNA-binding specificity of Ss-LrpB and the elaboration of an energy normalized sequence logo. The latter is a valuable tool for the search of new binding sites and thus Ss-LrpB regulon members in the genome of S. solfataricus P2. Several promising potential targets of the regulator have already been identified, including a porDAB operon, encoding a pyruvate ferredoxine oxidoreductase, a potential transporter gene, a methylthioadenosine phosphorylase gene and a conserved gene of unknown function. Finally, we also obtained crystals of the C-terminal domain of Ss-LrpB, which is predicted to be a αβ-sandwich (βαββαβ-fold). This domain, also called Regulation of Amino acid Metabolism (RAM) domain, is responsible for oligomerization and ligand binding in other Lrp-like regulators. A data set was collected at 2 Å resolution.