We characterize in real time the composition and catalytic state of the initial Escherichia coli transcription-coupled repair (TCR) machinery by using correlative single-molecule methods. TCR initiates when RNA polymerase (RNAP) stalled by a lesion is displaced by the Mfd DNA translocase, thus giving repair components access to the damage. We previously used DNA nanomanipulation to obtain a nanomechanical readout of protein-DNA interactions during TCR initiation. Here we correlate this signal with simultaneous single-molecule fluorescence imaging of labeled components (RNAP, Mfd or RNA) to monitor the composition and localization of the complex. Displacement of stalled RNAP by Mfd results in loss of nascent RNA but not of RNAP, which remains associated with Mfd as a long-lived complex on the DNA. This complex translocates at ∼4 bp/s along the DNA, in a manner determined by the orientation of the stalled RNAP on the DNA.
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We thank N. Savery, S. Darst, R. Landick and J. Gelles for stimulating conversations, members of the Strick laboratory for critical discussions, N. Joly (Institut Jacques Monod) for a generous gift of Mfd and W. Grange (University of Paris Diderot) for cloning of the AviTag into the β′ subunit of RNAP. This work was made possible by a European Science Foundation (EURYI) grant, European Union Seventh Framework Program grant (HEALTH-F4-2008-223545) and a French Agence National de la Recherche grant (RepOne) to T.R.S., in addition to support from the Fondation Fourmentin-Guilbert, and CNRS and University of Paris Diderot core funding.
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 Supplemental correlative nanomanipulation-fluorescence traces probing nascent RNA and fluorescent Mfd during TCR initiation.
Supplemental correlative nanomanipulation-fluorescence traces showing (a) loss of fluorescent DNA probe signal upon formation of the Mfd-RNAP repair intermediate and (b) increase in fluorescence signal of SNAP-Mfd over the lifetime of the intermediate. In one of the latter traces photobleaching is observed prior to resolution of the intermediate, but not before a marked increase in fluorescence of SNAP-Mfd can be recorded. In another of the traces in this series, the DY-549 fluorescence of labeled Mfd appears approximately 30s prior to formation of the intermediate, consistent with the ~20 s rate-limiting catalytic step identified for this system13.
Supplementary Figure 2 Supplemental correlative nanomanipulation-fluorescence traces probing fluorescent RNAP during TCR initiation.
Supplemental correlative nanomanipulation-fluorescence traces showing an increase or a decrease, respectively, in fluorescence of SNAP-RNAP during the lifetime of the intermediate when RNAP was originally directed to transcribe (a) towards or (b) away from the surface. Traces for which there is no break in the time-axis were obtained by monitoring the nanomanipulated DNA for fluorescence even absent RNAP, and here the moment of binding of RNAP is seen to be coincident with formation of the initially-transcribing complex. Traces for which there is a break in the time-axis were obtained via pulse-chase experiments where RNAP was first seen to load onto DNA in the mechanical channel, and then excess RNAP washed out (break) before fluorescence imaging was started.
Supplementary Figure 3 Supplemental correlative nanomanipulation-fluorescence traces probing fluorescent RNAP transcribing towards the surface.
Nanomanipulation and fluorescence time-traces for transcription, directed towards the surface, of the 400 bp construct (TTS400, see Supplementary Note). Such a transcribed region represents 140 nm of DNA contour length, but only 90 nm of vertical rise through the TIR field at the low extending force (F=0.3 pN) used here. (a) Time-traces corresponding to ~2/3 of the optimal events (11/15) described in the Venn diagram (Supp. Fig. 4) [The remaining 4 events were nanomechanically validated and displayed fluorescence but presented no increase in fluorescence for the duration of the event and were not fitted are are not shown]. When a clear change in fluorescence signal is observed (SNR>2.5 at RNAP binding and followed by fluorescence increasing by at least a factor of two during elongation) it is possible to obtain an estimate of velocity which is in reasonable agreement with the nanomechanical signal. Thus in red are shown single-exponential fits to the fluorescence increase in the time interval between promoter escape and transcription termination (as determined from the nanomechanical channel, vertical grey guides). The time inscribed above the nanomechanical trace gives this time interval. The time inscribed in the fluorescence trace represents the time constant for a single-exponential rise in fluorsecence (red line) obtained via non-linear fitting of the background-corrected data with fitting error bars obtained from photon counting statistics. Because the TIR depth in this experiment was ~120 nm (below) and the vertical rise in the TIR field during elongation is ~90 nm, this time has been corrected by 25% before being inscribed here; it therefore represents the time required for transcription of 400 bp as estimated from the exponential properties of the TIR field traversed by the fluorescent RNAP. (b) Calibration of the TIR field for these experiments gave a good fit to a single exponential with decay length of 118 +/- 7 nm (s.e.m). Error bars on the fluorescence signal represent the standard error on the mean for counting statistics; error bars on the extension signal are negligeable. We note that the decay length, λ, thus obtained is consistent with expectations based on the equation λ = λin/4π[(n1sin θ1)2 – (n2)2]1/2, where λin is the input wavelength, n1 the index of refraction of the glass, θ1the angle of the incoming beam relative to the optical axis, and n2 the index of refraction of the media in which the evanescent wave decays. For λin = 532 nm, n1 = 1.52, q = 68o (calculated for the input beam displaced from the optical axis by 4.2 mm, using the fact that in this setup first TIR light is observed at the glass-water interface for a displacement of 3.1 mm from the optical axis) and n2 = 1.33, we obtain l ~ 90 nm, in reasonable agreement with the value obtained experimentally. Deviation from ideal behavior may come from both extrinsic effects (eg an imperfectly focused input beam may cause light to enter the objective at slightly different effective NAs) and intrinsic effects (eg if the magnetic beads were to display nonlinear autofluorescence). (c) The distribution of RDe lifetimes from mechanical events (corresponding to the time taken to transcribe 400 bp and terminate, and for which the termination time is small compared to the elongation time35). These events correspond to the uncorrupted nanomechanical traces of transcription obtained for this dataset (ie the 85 events depicted in the Venn diagram Supplementary Figure 4). A gaussian fit to the data gives a lifetime of 40 +/- 1 s. (standard deviation 9 bp/s), corresponding to a velocity of 10 bp/s, in agreement with rates expected for NTP concentrations (200 μM) and temperature (T=28oC) used here.
Venn diagrams of events obtained and presented. The surface of the white box represents the total number (n) of nanomechanical events obtained for each condition. Numbers inscribed within the diagram specify the number of events represented by the delineated surface they are contained within. The surface of the yellow oval represents the number of nanomechanical events for which a flawless nanomechanical trace was obtained over the entire duration of the reaction – i.e. for which 1) there are clearly-detectable transitions in the DNA extension corresponding to well-established transcription events, 2) there is no excessive mechanical drift or noise over ~20-30 minutes, and 3) the intermediate state of a first complex is not interrupted by stochastic loading of a second RNAP13. This represents typically upwards of 2/3 of experiments. The surface of the pink oval represents the number of nanomechanical events for which single-molecule fluorescence is concomittantly monitored flawlessly over the lifetime of the object of interest – ie for which 1) the protein or probe turns out to indeed have a functionning fluorophore attached to it, 2) there is no premature photobleaching and 3) fluorescence is not altered by loading of a second fluorecent RNAP. The pink region of this oval and the number inscribed therein corresponds to flawless fluorecent events but corrupted nanomechanical events. The orange region of overlap between yellow and pink ovals corresponds to events presented in this paper, i.e. those events for which there is full monitoring of the process both in the nanomechanical channel and in the fluorescence channel. At the same time all of the data extracted from the partial or interrupted events not part of this orange overlap supports the observations based on data with full coverage in both mechanical and fluorescence channels.
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Graves, E., Duboc, C., Fan, J. et al. A dynamic DNA-repair complex observed by correlative single-molecule nanomanipulation and fluorescence. Nat Struct Mol Biol 22, 452–457 (2015). https://doi.org/10.1038/nsmb.3019
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