Characterisation of the TATA-box binding protein (TBP) from the Antarctic archaeon, Methanococcoides burtonii

• Main Author: Chong, Wai Yin Kevin
• Format: Doctoral or Postdoctoral Thesis
• Language: English
• Published: UNSW, Sydney 2011
• Subjects: Methanococcoides burtonii; Archaea; Cold adaption; Transcription initiation; TATA-box binding protein (TBP); MbTBP206; MbTBP183; Isoforms; N-terminal repeat; Biophysical and functional characterisation; Promoter-binding; Antarctic; The Antarctic; Antarc*
• Online Access: http://hdl.handle.net/1959.4/50946; https://unsworks.unsw.edu.au/bitstreams/ebe93876-8526-448f-8630-ba90b7963bb5/download; https://doi.org/10.26190/unsworks/23753

The TATA-box binding protein (TBP) plays an important role in transcription initiation. TBPs from many eucaryotes and archaea have been studied. However the structure and function of TBP from psychrophilic (cold-adapted) organisms is less understood. Methanococcoides burtonii is a psychrophilic archaeon that has served well for studies of cold adaption in archaea. The complete genome sequence for M. burtonii is published, and a single tbp gene was identified. Translation of the M. burtonii tbp open reading frame was found to occur from two separate AUG codons, producing TBP isoforms containing 206 (MbTBP206) and 183 (MbTBP183) amino acids. Additionally, MbTBP206 contains a sequence repeat (RepA) in the N-terminal region that is absent in MbTBP183. Both isoforms possess identical putative DNA-interacting residues and are predicted to form a saddle-like conformation, typical of TBPs. Both isoforms were detected in vivo by Western blotting, and found to be over-abundant at 4°C than 23°C. Recombinant MbTBP isoforms produced were shown by gel shift assays to preferentially interact with oligonucleotides containing TATA-box sequences; a functional trait typical of TBPs. Biophysical characterisation of both recombinant isoforms included evaluation of secondary structure and protein hydrophobicity by circular dichroism (CD) and ANSfluorescence, respectively. Both isoforms were found to contain secondary structures typical of TBPs, with an unstructured RepA region in MbTBP206. Hydrophobicity studies also implied that MbTBP206 was more thermostable than MbTBP183. Further, heat-induced loss of protein conformation in both recombinant MbTBP from 4°C to 28°C was reduced by the addition of glutamate and aspartate, both of which are previously found intracellular solutes of M. burtonii. As such, functional characterisations of both isoforms were conducted using buffers that mimic the intracellular milieu of M. burtonii. In order to elucidate functional differences between the isoforms and begin to determine putative binding ...