The role of TATA binding protein and other factors in determining promoter strength

Abstract

Many eukaryotic RNA polymerase II core promoters contain a TATA box with the consensus sequence TATAAA, which is the recognition site for the TATA-binding protein (TBP). There are also a large number of functional core promoters that contain sequences that differ from the consensus element, but these promoters still depend on TBP for transcription initiation. To determine the mechanistic differences in transcription initiation between TATAAA and non-consensus sequences, we compared the functional activity of thirteen TATA elements, each with a systematic replacement of a cytosine or a guanine base at each of the six positions in the TATAAA sequence. Each of the single base substitutions in the consensus TATA sequence leads to severe transcriptional defects in vivo and disruption of various protein-DNA complexes in vitro, depending on the position involved. Initial binding of TBP, TFIIA-TBP and TFIIB-TBP to the different elements revealed that single base replacements in the first position (CATAAA) or the last position (TATAAG), did not affect the initial formation of any of these complexes, although transcription is severely compromised in vivo. Strikingly, the TFIIA-TBP-DNA complex was significantly less stable on the CATAAA and TATAAG elements when compared to the TATAAA element. This loss of stability was specific to the higher order complex containing TFIIA since there was little difference in the stability of TBP-DNA or TFIIB-TBP-DNA complexes. However, when the N-terminal domain of TBP was removed, the TFIIA-TBP complex could no longer distinguish between TATAAA and CATAAA, suggesting a role for this domain of TBP in determining promoter strength. Taken together, these results suggest that TBP binding to the core promoter and interacting with other components of the general transcription machinery, especially TFIIA, are important considerations for RNA pol II transcription regulation.