Abstract:
:Here we describe the application of a novel combinatorial method, restriction endonuclease protection selection and amplification (REPSA), to identification of a consensus DNA binding site for the TATA binding subunit (hTBP) of the human general transcription factor TFIID. Unlike most combinatorial methods, REPSA is based on inhibition of an enzymatic template inactivation process by specific ligand-DNA complexes. The mild conditions of this method allow examination of proteins with atypical binding characteristics (e.g. limited discrimination between specific and non-specific binding sites), such as those found with hTBP. Analysis of 57 emergent sequences identified 47 sequences containing consensus 6 bp TATA elements as previously defined. However, further examination of these sequences indicated that a larger consensus, 5'-TATAAATA-3', could be supported by the data. Studies of the binding affinities and transcriptional activities of these four consensus TATA sequences demonstrated that hTBP binding affinity correlated directly with transcriptional activity in vitro and that the TATAAATA sequence was the best among the TATA sequences investigated.
journal_name
Nucleic Acids Resjournal_title
Nucleic acids researchauthors
Hardenbol P,Wang JC,Van Dyke MWdoi
10.1093/nar/25.16.3339subject
Has Abstractpub_date
1997-08-15 00:00:00pages
3339-44issue
16eissn
0305-1048issn
1362-4962pii
gka522journal_volume
25pub_type
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