Doctor of Philosophy (PhD)
The TATA-binding protein (TBP) is required for transcription by all three nuclear RNA polymerase systems. TBP binds the TATA box (consensus sequence TATAa/tAa/tN) and induces an ~80° bend in the DNA. To achieve recruitment of Saccharomyces cerevisiae RNA polymerase III, TBP is associated with two additional factors, Brf1 and Bdp1, to form initiation factor TFIIIB. This study focuses on interactions between the proteins and DNA in TFIIIB, and effects of promoter topology on transcription. Previous data suggests that the structure or dynamics of the TBP-DNA complex may be altered upon entry of Brf1 and Bdp1 into the complex. Here, an altered specificity TBP mutant TBPm3 and iterative in vitro selection assays are used to show that Brf1 and Bdp1 impose strict sequence preference on TBPm3 for the downstream half of the TATA box. Notably, the selected sequence (TGTAAATA) perfectly matches the TATA box of the pol III-transcribed U6 small nuclear RNA (SNR6) gene, suggesting that the selected T•A base pair step at the downstream end of the 8 bp TBP site may provide a DNA flexure that promotes TFIIIB-DNA complex formation. Increased TBP-induced bend angle has been correlated with increased levels of relative pol II transcription from DNA with variant TATA boxes. Here, circular permutation and electrophoretic mobility shift assays are used to show that, in a pol III system, TATA box sequence has almost no effect on protein-induced bend angle. In vitro transcription is used to show that, despite the lack of difference observed in TBP-induced bend angles, differences in relative transcription distinct from those observed in a pol II system occur; therefore, it may be that flexure or dynamics of the TATA box sequence governs transcription efficiency. Notably, despite presence of a native pol III composed of 7 consecutive thymines terminator, termination is unexpectedly inefficient. Since the templates contain only five base pairs of pol III gene sequence beyond the terminator, these data suggest that sequence well beyond the terminator may affect proper termination of transcription, and that termination by pol III requires more than the well-characterized string of thymines.
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Tsihlis, Nick Dimitrios, "Investigation of Protein-DNA Contacts in the Saccharomyces Cerevisiae RNA Polymerase III Recruitment Factor (TFIIIB) Complex and Effects of Promoter Topology on Transcription" (2006). LSU Doctoral Dissertations. 2060.