Interestingly, the addition of the third initiation factor, Bdp1, yields an outstandingly stable initiation complex at the U6 promoter. This in turn requires robust formation of initiation complexes at the promoter as transcriptional regulation cannot take place at the level of elongation at these extremely short genes. Hence, TFIIIB is likely to experience mechanical forces that are compensated by the extremely stable initiation complex. Moreover, in a model where the polymerase remains bound to the promoter, strain would build up during transcription between the promoter binding site and the active site due to the increasing amount of transcribed DNA that has to be accommodated in the polymerase.
This additionally increases the forces that the transcription initiation complex has to withstand. In this case, initiation factors need to be stably attached at the promoter in order to avoid displacement by the nucleosome. This indicates that the tension on the DNA might be a mechanism of gene regulation.
The packaging, histone placement, action of the replication machinery and binding of regulatory proteins will certainly have an impact on the tension that the initiation complex is exposed to. Thus, besides steric effects, tension influences transcription. On the other hand, after the transcription initiation complex has formed i. This might indicate that after the decision for transcription was taken, the process should become independent of mechanical factors ensuring that the RNAP enters the elongation phase of transcription.
All proteins were expressed and purified as described previously 57 , Cloning was performed using the replicative form dsDNA of the M13 phage. Phage ssDNA production was carried out in E. Five millilitres of PPB2 lysis buffer 0. The final DNA strand carries both dyes and is complementary to the promoter region of the origami scaffold. To this end, RNA loading buffer The gel was visualised under UV light and the band corresponding to the doubly labelled DNA strand was excised and pulverised.
DNA origamis were assembled as described previously 63 see Supplementary Note 1. All purification steps were repeated once. A total of 94 micrographs were semi-automatically collected at 40, magnification 2.
Contrast transfer function CTF estimation and manual particle picking were carried out in Relion 3 Silica microscope slides used for TIRF experiments were prepared as described before Flow chambers were prepared and assembled as described before For fluorescence measurements, the flow chamber was incubated with 0.
Time-resolved single-molecule fluorescence measurements were performed on a homebuilt prism-type total internal reflection setup based on a Leica DMi8 inverse research microscope. The videos were analysed employing the iSMS software package 84 using the programs defaults settings. FRET efficiencies were calculated as proximity ratios from fluorescence intensity—time traces that were corrected for background fluorescence using the average intensity of all pixels with a 2 pixel distance to the molecule spot.
The FRET efficiency E and the stoichiometry factor S describing the relative ratio between donor and acceptor signal for each molecule were calculated as:. FRET efficiency histograms were calculated from all frames of traces showing dynamic switching between states with a stoichiometry value between 0. The histograms of at least three independent experiments were normalised and fitted with a mono-exponential decay function to calculate the mean dwell time in the high FRET state TBP bound to DNA.
For equilibrium measurements Figs. For time-course experiments Fig. Emitted fluorescence was collected using a 1. A dichroic mirror Tlpxr; Chroma separated donor and acceptor fluorescence. Burst data were corrected for donor leakage and direct excitation of the acceptor determined from APBS according to ref. Therefore, we assume that this effect has no connection to the biological system. To account for this density during data analysis, we added an additional Gaussian with a fixed area to all our fits to improve the overall fit quality see Supplementary Table 2 , but did not include it in the Results and Discussion sections.
Data were processed as above. Low FRET and high FRET bins were normalised to the combined sum to determine relative ratios of both populations, which were plotted against time and fitted with a mono-exponential function. All factors except TBP were mixed prior to data acquisition.
This decay constant translates to the complex assembly rate that is superimposed with the simultaneous complex disassembly. We used a perturbation-relaxation kinetics model to extract both rates from the data. The equilibrium constant K is given by the ratio of the rates:.
The system is perturbed by a change in the TBP concentration and will relax in a first-order process to its new equilibrium. Since k bent is concentration dependent, the unbent fraction [ u ] will vary according to:. Fluorescence correlation spectroscopy analysis was performed with the PicoQuant software package SymPhoTime The relative diffusion times given in Supplementary Fig.
The statistical error shown is the standard error of the fit. Further information on research design is available in the Nature Research Reporting Summary linked to this article.
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Talemi, R. C 73, — Article Contents Abstract. Binding of DNA origami to lipids: maximizing yield and switching via strand displacement.
School of Chemistry, The University of Sydney. Oxford Academic. Google Scholar. Esther Darley. Pietro Ridone. James P Gaston. Ali Abbas. Shelley F J Wickham. Correspondence may also be addressed to Shelley F. Email: shelley. Matthew A B Baker. To whom correspondence should be addressed.
Email: matthew. Revision received:. Select Format Select format. Permissions Icon Permissions. Abstract Liposomes are widely used as synthetic analogues of cell membranes and for drug delivery.
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