Identification of preferred hTBP DNA binding sites by the combinatorial method REPSA.

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 Res

journal_title

Nucleic acids research

authors

Hardenbol P,Wang JC,Van Dyke MW

doi

10.1093/nar/25.16.3339

subject

Has Abstract

pub_date

1997-08-15 00:00:00

pages

3339-44

issue

16

eissn

0305-1048

issn

1362-4962

pii

gka522

journal_volume

25

pub_type

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