Single-Molecule Studies of Human RNA Polymerase Ii Transcription Initiation: Core-Promoter Recognition

Abstract

We have developed a robust experimental platform that allows us to study the assembly dynamics of complex, multi-components biochemical systems at the single-molecule level. A key element of our implementation was the refinement of surface passivation protocols for single-molecule experiments, to achieve a 10- to 30-fold reduction in non-specific interactions for the various components of the Human RNA Polymerase II machinery. The reduced background of this optimized system allowed us to monitor the assembly of fluorescent transcription machinery components on promoter-containing DNA molecules, using the mutli-color (sub-)nanometer co-localization capabilities of our recently developed actively-stabilized microscope setup (Pertsinidis et al., Nature 2010).In addition, we applied single-molecule spectroscopy tools, and as well as numerical modeling, to start dissecting the cascade of conformational rearrangements during Pre-initiation Complex (PIC) formation. Comprised of the General Transcription Factors (GTFs) TFIID/A/B/F/E/H and RNAPol II, assembly of this g50-polypeptide, Mega-Dalton complex commences with recognition of core-promoter elements by the TFIID complex. We observe that the TATA-binding (TBP) sub-unit of TFIID binds the consensus AdMLP core promoter with a transition to the severely kinked/partially unwound TATA-box state. Interestingly, we observe that TBP can also recognize variant TATA elements, resulting in structures with intermediate degrees of DNA deformation. Finally, our observations reveal additional conformational changes upon TFIIA (and to some extent TFIIB) recruitment, suggesting a mechanism for regulating further progression of the PIC assembly on strong as well as weak TATA-containing promoters but not on random DNA sequences.