The general transcription factors (GTFs) of eukaryotic RNA polymerase II, in a process facilitated by regulatory and accessory factors, target promoters through synergistic interactions with core elements. The specific binding of the TATA box-binding protein (TBP) to the TATA box has led to the assumption that GTFs recognize promoters directly, producing a preinitiation complex at a defined position. Using biochemical analysis as well as biophysical single-pair Förster resonance energy transfer, we now provide evidence that negative cofactor-2 (NC2) induces dynamic conformational changes in the TBP-DNA complex that allow it to escape and return to TATA-binding mode. This can lead to movement of TBP along the DNA away from TATA.

Polyamidoamine (PAMAM) dendrimers were modified and tested for use as solution-phase diffusion probes in silica nanostructures. In order for the successful application of dendrimers as solution-phase probes, their interactions with silica surfaces must be understood and controlled, so that the motion of the probe is not influenced by adsorption. Adsorption/desorption kinetics of PAMAM dendrimers and their diffusion in solution near silica surfaces were investigated with total internal reflection fluorescence correlation spectroscopy (TIR-FCS). Dendrimers of generations 3, 5, and 7 were dye-labeled with carboxyrhodamine 6G. Because PAMAM dendrimers are positively charged in solution (having primary amines as end groups), significant adsorption of these molecules to the negatively charged silica surface was observed. Adsorption/desorption rates and the equilibrium constant for adsorption were determined by fitting the autocorrelation functions to a kinetic model. The desorption rate decreases and the absorption equilibrium constant increases with higher dendrimer generation. To reduce the adsorption of these probes to silica surfaces, the labeled dendrimers were reacted with succinic anhydride, converting the primary amine end groups to negatively charged carboxylic acid groups. These carboxylated dendrimers did not detectably adsorb to silica from aqueous solution. TIR-FCS was used to determine their free-solution diffusion constants near silica surfaces, and the corresponding hydrodynamic radii compare favorably with values reported from forced Rayleigh scattering measurements.

Three generations of poly(amidoamine) dendrimers were dye-labeled and chemically modified to have terminal carboxyl groups and used as variably sized probes to study diffusion in thin sol-gel films. Total internal reflection fluorescence spectroscopy experiments, both correlation and concentration-jump measurements, were employed to measure the relative populations and effective diffusion coefficients of dendrimers in the films. For films prepared from small (27-nm) silica particles, larger dendrimers could be completely excluded from penetrating the sol-gel structure. In films made of larger (150-nm) particles with correspondingly larger pores, concentration-jump experiments showed that larger dendrimers are excluded from more of the intraparticle pore space than small dendrimers. Similarly, fluorescence-correlation measurements showed that the diffusion of smaller dendrimers exhibited greater tortuosity than larger dendrimers in the interparticle pores of the film. The smaller dendrimers explore a greater volume of smaller, more convoluted pores, whereas larger dendrimers penetrate a smaller volume of larger, more open pores.

Single-pair Fö rster resonance energy transfer (spFRET) has become an important tool for investigating conformational dynamics in biological systems. To extract dynamic information from the spFRET traces measured with total internal reflection fluorescence microscopy, we extended the hidden Markov model (HMM) approach. In our extended HMM analysis, we incorporated the photon-shot noise from camera-based systems into the HMM. Thus, the variance in Fö rster resonance energy transfer (FRET) efficiency of the various states, which is typically a fitted parameter, is explicitly included in the analysis estimated from the number of detected photons. It is also possible to include an additional broadening of the FRET state, which would then only reflect the inherent flexibility of the dynamic biological systems. This approach is useful when comparing the dynamics of individual molecules for which the total intensities vary significantly. We used spFRET with the extended HMM analysis to investigate the dynamics of TATA-box-binding protein (TBP) on promoter DNA in the presence of negative cofactor 2 (NC2). We compared the dynamics of two promoters as well as DNAs of different length and labeling location. For the adenovirus major late promoter, four FRET states were observed; three states correspond to different conformations of the DNA in the TBP-DNA-NC2 complex and a four-state model in which the complex has shifted along the DNA. The HMM analysis revealed that the states are connected via a linear, four-well model. For the H2B promoter, more complex dynamics were observed. By clustering the FRET states detected with the HMM analysis, we could compare the general dynamics observed for the two promoter sequences. We observed that the dynamics from a stretched DNA conformation to a bent conformation for the two promoters were similar, whereas the bent conformation of the TBP-DNA-NC2 complex for the H2B promoter is approximately three times more stable than for the adenovirus major late promoter.