Comparison of TATA-binding Protein Recognition of a Variant and Consensus DNA Promoters

Robyn M Powell,Kay M Parkhurst,Lawrence J Parkhurst

doi:10.1074/jbc.m110147200

Abstract

Assembly of transcription pre-initiation complexes proceeds from the initial complex formed between "TATA" bearing promoter DNA and the TATA-binding protein (TBP). Our laboratory has been investigating the relationships among TATA sequence, TBP center dot TATA solution structure, recognition mechanisms, and transcription efficiency. TBP center dot TATA interactions have been modeled by global analysis of detailed kinetic and thermodynamic data obtained using fluorimetric and fluorometric techniques in conjunction with fluorescence resonance energy transfer. We have reported recently that TBP recognition of two consensus promoters, adenovirus major late (AdMLP: TATAAAAG) and E4 (TATATATA), is well described by a linear two-intermediate mechanism with simultaneous DNA binding and bending. Similar DNA geometries and high transcription efficiencies characterize these TBP x TATA complexes. Here we show that, in contrast to the consensus sequences, TBP recognition of a variant sequence (C7: TATAAACG) is described by a three-step model with two branching pathways. One pathway proceeds through an intermediate having severely bent DNA, reminiscent of the consensus interactions, with the other branch yielding a unique conformer with shallowly bent DNA. The resulting TBP x C7 complex has a dramatically different solution conformation than for TBP x DNA(CONSENSUS) and is correlated with diminished relative transcription activity. The temperature dependence of the TBP x C7 helical bend is postulated to derive from population shifts between the conformers with slightly and severely bent DNA.

Highlights

Our laboratory has been studying the detailed recognition mechanism of DNA promoters by S. cerevisiae TATA-binding protein (TBP) [15, 16, 20] and the solution geometries of the resulting TBP1⁄7TATA complexes [34, 35]
Our laboratory has been investigating the relationships among TATA sequence, TBP1⁄7TATA solution structure, recognition mechanisms, and transcription efficiency
TBP1⁄7TATA interactions have been modeled by global analysis of detailed kinetic and thermodynamic data obtained using fluorimetric and fluorometric techniques in conjunction with fluorescence resonance energy transfer

Summary

Introduction

Our laboratory has been studying the detailed recognition mechanism of DNA promoters by S. cerevisiae TBP [15, 16, 20] and the solution geometries of the resulting TBP1⁄7TATA complexes [34, 35]. Global analysis of extensive real-time kinetic and thermodynamic data sets first revealed a linear three-step mechanism for the TBP1⁄7AdMLP reaction [16], with intermediate conformers having DNA bent to the same extent as in the final complex. These conformers are present at high mole fraction throughout the reaction and persist at equilibrium. TBP-bound C7 has a helical bend dramatically different from that in bound AdMLP that correlates with significantly reduced relative transcription efficiency [34] This solution geometry is highly sensitive to the presence and concentration of osmolytes [35], in contrast to that for the complexes bearing consensus sequences. The temperature dependence of the TBP-bound C7 solution bend angle is proposed to arise from population shifts between conformers with slightly and severely bent DNA within a two-state model

Results

Discussion

Conclusion