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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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MPE-Fe(II) 2D footprinting confirms binding of TBPm3 at the TGTA box. Densitometry profiles of 76 bp DNA containing the favored sequence (TGTAAATTG) incubated with (black line) and without (blue line) TBPm3 show protection at the TGTA box. Numbering is based on the start site of transcription (+1). Gray line represents uncut DNA.
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fig5: MPE-Fe(II) 2D footprinting confirms binding of TBPm3 at the TGTA box. Densitometry profiles of 76 bp DNA containing the favored sequence (TGTAAATTG) incubated with (black line) and without (blue line) TBPm3 show protection at the TGTA box. Numbering is based on the start site of transcription (+1). Gray line represents uncut DNA.

Mentions: The results of the TBPm3 selection were verified by EMSA and MPE-Fe(II) footprinting on a DNA probe representing the most frequently selected bases at each position, TGTAAATTG (note that this sequence represents the most frequently occurring bases at each randomized position, but was not found among the selected clones). TBPm3 was seen to bind to 26 bp DNA containing this sequence, while disfavored sequences, containing for example a C in position N1 (TGTAACTGG) yield barely detectable complex formation (Figure 4 and data not shown). MPE-Fe(II) footprinting on the 76 bp DNA containing the favored sequence indicates that TBPm3 is binding at the TGTA box, despite the fact that this sequence was not found among the selected clones, as seen by the partial protection from cleavage at positions −28 to −23 (Figure 5), where the first T of the TGTA box is designated −30. Enhanced cleavage was observed at base pair −19, −18 and −31 consistent with that observed for wild-type TBP at these positions (22,29).


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)

MPE-Fe(II) 2D footprinting confirms binding of TBPm3 at the TGTA box. Densitometry profiles of 76 bp DNA containing the favored sequence (TGTAAATTG) incubated with (black line) and without (blue line) TBPm3 show protection at the TGTA box. Numbering is based on the start site of transcription (+1). Gray line represents uncut DNA.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: MPE-Fe(II) 2D footprinting confirms binding of TBPm3 at the TGTA box. Densitometry profiles of 76 bp DNA containing the favored sequence (TGTAAATTG) incubated with (black line) and without (blue line) TBPm3 show protection at the TGTA box. Numbering is based on the start site of transcription (+1). Gray line represents uncut DNA.
Mentions: The results of the TBPm3 selection were verified by EMSA and MPE-Fe(II) footprinting on a DNA probe representing the most frequently selected bases at each position, TGTAAATTG (note that this sequence represents the most frequently occurring bases at each randomized position, but was not found among the selected clones). TBPm3 was seen to bind to 26 bp DNA containing this sequence, while disfavored sequences, containing for example a C in position N1 (TGTAACTGG) yield barely detectable complex formation (Figure 4 and data not shown). MPE-Fe(II) footprinting on the 76 bp DNA containing the favored sequence indicates that TBPm3 is binding at the TGTA box, despite the fact that this sequence was not found among the selected clones, as seen by the partial protection from cleavage at positions −28 to −23 (Figure 5), where the first T of the TGTA box is designated −30. Enhanced cleavage was observed at base pair −19, −18 and −31 consistent with that observed for wild-type TBP at these positions (22,29).

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