Affinity, stability and polarity of binding of the TATA binding protein governed by flexure at the TATA Box.
The TATA binding protein (TBP), which plays a central role in gene regulation as an essential component of all three nuclear transcription systems, sharply kinks the TATA box at two sites and severely contorts the intervening DNA segment. DNA constructs with precisely localized flexure have been used to investigate the special repertoire of mechanisms and properties that arise from TBP interacting with the TATA box. DNA flexure precisely localized to the sites of TBP-mediated DNA kinking increases the affinity of TBP more than 100-fold; unexpectedly, this increase in affinity is achieved almost exclusively by increasing the stability of the TBP-DNA complex rather than the rate of its formation. In vitro transcription with RNA polymerase III provides a first demonstration that the orientation of TBP on the TATA box is governed by DNA deformability, its C-proximal repeat contacting the more flexible end of the TATA box. Exceptionally stable TBP-DNA complexes reach their orientational equilibrium very slowly; in these circumstances, assembly of stable ("committed") transcription initiation complexes can freeze far-from-equilibrium orientations of TBP on the TATA box, causing transcription polarity to be determined by a kinetic trapping mechanism.
Grove
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A
Department of Biology and Center for Molecular Genetics, University of California, 9500 Gilman Drive, San Diego, CA, 92093-0634, USA.
Galeone
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A
Yu
E
E
Mayol
L
L
Geiduschek
E P
EP
D020029:Base Pairing; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D006207:Half-Life; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D010432:Pentoxyl / Q000031:analogs & derivatives / Q000378:metabolism; D012320:RNA Polymerase III / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D016385:TATA Box / Q000235:genetics*; D035181:TATA-Box Binding Protein; D013698:Templates, Genetic; D013816:Thermodynamics; D035361:Transcription Factor TFIIIB; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D014158:Transcription, Genetic / Q000235:genetics
D004268:DNA-Binding Proteins; D035181:TATA-Box Binding Protein; D035361:Transcription Factor TFIIIB; D014157:Transcription Factors; C008055:5-hydroxymethyluracil; D010432:Pentoxyl; D004247:DNA; D012320:RNA Polymerase III