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The Saccharomyces cerevisiae RNA polymerase III recruitment factor subunits Brf1 and Bdp1 impose a strict sequence preference for the downstream half of the TATA box.

Tsihlis ND, Grove A - Nucleic Acids Res. (2006)

Bottom Line: To achieve recruitment of Saccharomyces cerevisiae RNA polymerase III, TBP is associated with two additional factors, Brf1 and Bdp1, to form the initiation factor TFIIIB.Notably, the selected sequence (TGTAAATA) is a perfect match to the TATA box of the RNA polymerase III-transcribed U6 small nuclear RNA (SNR6) gene.We suggest 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803, USA.

ABSTRACT
Association of the TATA-binding protein (TBP) with its cognate site within eukaryotic promoters is key to accurate and efficient transcriptional initiation. To achieve recruitment of Saccharomyces cerevisiae RNA polymerase III, TBP is associated with two additional factors, Brf1 and Bdp1, to form the initiation factor TFIIIB. Previous data have suggested that the structure or dynamics of the TBP-DNA complex may be altered upon entry of Brf1 and Bdp1 into the complex. We show here, using the altered specificity TBP mutant TBPm3 and an iterative in vitro selection assay, that entry of Brf1 and Bdp1 into the complex imposes a strict sequence preference for the downstream half of the TATA box. Notably, the selected sequence (TGTAAATA) is a perfect match to the TATA box of the RNA polymerase III-transcribed U6 small nuclear RNA (SNR6) gene. We suggest 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.

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Alignment of sequences selected by TFIIIB. Only sequences retaining the original TGTAA sequence are shown. Bases corresponding to randomized positions are in boldface.
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fig6: Alignment of sequences selected by TFIIIB. Only sequences retaining the original TGTAA sequence are shown. Bases corresponding to randomized positions are in boldface.

Mentions: When the selection was performed using TFIIIB assembled with TBPm3, a distinct sequence preference emerged. A total of 25 clones containing the TGTAA sequence were aligned to determine the consensus for this selection. As for the TBPm3 selection, clones containing the TATAA sequence (25 clones) were excluded from the alignment, as were 13 clones containing the series of GTG repeats. While the occurrence of the GTG-repeat sequences in both selections is curious, we did not pursue this observation further. The remainder of the clones contained sequence with no match to the above categories, including other alterations to the 5′ half of the TGTA box. Alignment of the 25 TGTAA-containing sequences (Table 3) showed a much stronger sequence preference compared with TBPm3 alone, despite reaction conditions that should have allowed less stringent binding (100 mM NaCl versus 150 mM for the TBPm3 selection; Figure 6). Notably, the selected consensus sequence (TGTAAATAG) is a perfect match to the 8 bp U6 TATA box (TATAAATA).


The Saccharomyces cerevisiae RNA polymerase III recruitment factor subunits Brf1 and Bdp1 impose a strict sequence preference for the downstream half of the TATA box.

Tsihlis ND, Grove A - Nucleic Acids Res. (2006)

Alignment of sequences selected by TFIIIB. Only sequences retaining the original TGTAA sequence are shown. Bases corresponding to randomized positions are in boldface.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1636458&req=5

fig6: Alignment of sequences selected by TFIIIB. Only sequences retaining the original TGTAA sequence are shown. Bases corresponding to randomized positions are in boldface.
Mentions: When the selection was performed using TFIIIB assembled with TBPm3, a distinct sequence preference emerged. A total of 25 clones containing the TGTAA sequence were aligned to determine the consensus for this selection. As for the TBPm3 selection, clones containing the TATAA sequence (25 clones) were excluded from the alignment, as were 13 clones containing the series of GTG repeats. While the occurrence of the GTG-repeat sequences in both selections is curious, we did not pursue this observation further. The remainder of the clones contained sequence with no match to the above categories, including other alterations to the 5′ half of the TGTA box. Alignment of the 25 TGTAA-containing sequences (Table 3) showed a much stronger sequence preference compared with TBPm3 alone, despite reaction conditions that should have allowed less stringent binding (100 mM NaCl versus 150 mM for the TBPm3 selection; Figure 6). Notably, the selected consensus sequence (TGTAAATAG) is a perfect match to the 8 bp U6 TATA box (TATAAATA).

Bottom Line: To achieve recruitment of Saccharomyces cerevisiae RNA polymerase III, TBP is associated with two additional factors, Brf1 and Bdp1, to form the initiation factor TFIIIB.Notably, the selected sequence (TGTAAATA) is a perfect match to the TATA box of the RNA polymerase III-transcribed U6 small nuclear RNA (SNR6) gene.We suggest 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803, USA.

ABSTRACT
Association of the TATA-binding protein (TBP) with its cognate site within eukaryotic promoters is key to accurate and efficient transcriptional initiation. To achieve recruitment of Saccharomyces cerevisiae RNA polymerase III, TBP is associated with two additional factors, Brf1 and Bdp1, to form the initiation factor TFIIIB. Previous data have suggested that the structure or dynamics of the TBP-DNA complex may be altered upon entry of Brf1 and Bdp1 into the complex. We show here, using the altered specificity TBP mutant TBPm3 and an iterative in vitro selection assay, that entry of Brf1 and Bdp1 into the complex imposes a strict sequence preference for the downstream half of the TATA box. Notably, the selected sequence (TGTAAATA) is a perfect match to the TATA box of the RNA polymerase III-transcribed U6 small nuclear RNA (SNR6) gene. We suggest 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.

Show MeSH