High resolution functional dissection of the 'B-Finger' domain of basal transcription factor TFB/TFIIB. (360G-Wellcome-078043_Z_05_Z)

The core archaeal transcriptional machinery consists of TATA-binding protein (TBP), TFB (a homolog of eukaryotic TFIIB) and RNA polymerase (RNAP). The work described in this proposal will focus on a detailed characterization of the functional properties of the archaeal basal transcription factors TFB. This factor cooperates intimately with RNA polymerase (RNAP) during promoter recruitment and transcript initiation (especially the abortive transcription stage). Based on previous work, we have developed a series of assays for testing a variety of subfunctions of TFB, which we will apply to understand the structural basis of these activities in more detail. Specifically, we will dissect by site-directed mutagenesis the function of the B-finger of TFB which is inserted into the immediate vicinity of the catalytic site of RNAP. A bioinformatics analysis of B-fingers shows a high degree of variability (especially in the electrostatic properties) across the evolutionary range which are likely to result in significant functional differences in e.g. start site selection and/or abortive transcription behaviour. Due to the structural similarities between the archaeal transcription system and the eukaryotic RNAPII machinery many of the insights that we will obtain will also be applicable to understanding gene expression mechanisms in higher organisms.