STRUCTURAL ANALYSIS OF THE TBP/TFIIB/DNA COMPLEX FROM THE HYPERTHERMOPHILIC ARCHAEA PYROCOCCUS WOESEI. P. Kosa, B. DeDecker, G. Ghosh, P. B. Sigler, Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06510

Eukaryotic transcription Factor IIB is necessary for basal or activated transcription. TFIIB forms a complex with TATA-binding protein (TBP) and DNA. The crystal structure of this complex has been solved. TFIIB has been shown to directly interact with TBP, TFIIF, and RNA polymerase II, and is proposed to be involved in mediating activated transcription through an interaction with activators.

Homologs of TBP and TFIIB have been found in several Archaea species, suggesting that Archaea have a eukaryotic like pol II transcriptional apparatus. Pyrococcus woesei, an archaea from deep sea thermal vents which exhibits a maximum growth temperature of 110deg.C, has an A/T rich, TATA-like promoter sequence. The amino acid sequence of PwTBP is 40% identical to that of all eukaryotic species, and the TFIIB homolog (TFB) is 30% identical to eukaryotic factors. The homology extends throughout both sequences and the domain architecture of both proteins appears conserved.

In order to extend our structural understanding of the transcriptional preinitiation complex, we have grown crystals of PwTFB in a complex with PwTBP and an archael TATA-box for x-ray analysis. We have generated an MIR map, and we are in the process of tracing and refining the structure of the complex, with data to 2.1Å. Analysis of the Archaea complex may provide insight into the evolution of the transcriptional apparatus, the mechanism of thermostability, and the specificity of assembly of the ternary complex.