TBP binding to the TATA box induces a specific downstream unwinding site that is targeted by pluramycin.
BACKGROUND
The TATA-binding protein (TBP) is one of the major components of the human TFIID multiprotein complex. It is important in directing the initiation of RNA transcription at a site immediately downstream of the TATA sequence (TATA box) found in many eukaryotic promoters. The crystal structure of TBP complexed with an oligonucleotide containing the TATA box revealed a protein with an approximate two-fold symmetry which apparently has symmetrical interactions with DNA. It is not known how an asymmetric effect involving downstream activation can be produced by an apparent symmetric complex. We set out to examine the state of DNA in the TBP-DNA complex using pluramycin, a small molecular weight probe of DNA accessibility.


RESULTS
Binding of TBP to the TATA box facilitates intercalation of pluramycin at a defined site immediately downstream of the TATA sequence through an apparent transient unwinding of the DNA. Pluramycin adducts are detected by the production of DNA strand breakage products upon heating. Incubation of pluramycin with the TBP-DNA complex facilitates the trapping of the specific complex by intercalation. Gel mobility shift and circularization assays reveal that the binding of pluramycin on the 3'-side of the TATA box region considerably stabilizes the TBP-DNA complex.


CONCLUSIONS
We propose that the TBP-DNA-pluramycin ternary complex is a 'specific' binding mode in which TBP and pluramycin make compensatory alterations in DNA, accounting for the improved stability of the ternary complex. We also propose a model of the ternary complex that explains the observed asymmetric effect of TBP binding to the TATA box.
Sun
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Drug Dynamics Institute, College of Pharmacy, University of Texas at Austin 78712-1074, USA.
Hurley
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D000617:Aminoglycosides; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001483:Base Sequence; D003500:Cyclization; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D018983:DNA Footprinting; D004265:DNA Helicases / Q000737:chemistry / Q000187:drug effects*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D003850:Deoxyribonuclease I; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007364:Intercalating Agents / Q000494:pharmacology; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009211:Myoglobin / Q000737:chemistry / Q000378:metabolism; D012313:RNA / Q000096:biosynthesis; D016385:TATA Box / Q000187:drug effects*; D035181:TATA-Box Binding Protein; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D014158:Transcription, Genetic / Q000235:genetics
D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D004268:DNA-Binding Proteins; D007364:Intercalating Agents; D009211:Myoglobin; D035181:TATA-Box Binding Protein; D014157:Transcription Factors; C003169:pluramycin; D012313:RNA; D004247:DNA; D003850:Deoxyribonuclease I; D004265:DNA Helicases