Direct observation of abortive initiation and promoter escape within single immobilized transcription complexes

Abstract

Using total-internal-reflection fluorescence microscopy equipped with alternating-laser excitation, we were able to detect abortive initiation and promoter escape within single immobilized transcription complexes. Our approach uses fluorescence resonance energy transfer to monitor distances between a fluorescent probe incorporated in RNA polymerase (RNAP) and a fluorescent probe incorporated in DNA. We observe small, but reproducible and abortive-product-length-dependent, decreases in distance between the RNAP leading edge and DNA downstream of RNAP upon abortive initiation, and we observe large decreases in distance upon promoter escape. Inspection of population distributions and single-molecule time traces for abortive initiation indicates that, at a consensus promoter, at saturating ribonucleoside triphosphate concentrations, abortive-product release is rate-limiting (i.e., abortive-product synthesis and RNAP-active-center forward translocation are fast, whereas abortive-product dissociation and RNAP-active-center reverse translocation are slow). The results obtained using this new methodology confirm and extend those obtained from diffusing single molecules, and pave the way for real-time, single-molecule observations of the transitions between various states of the transcription complex throughout transcription.

FIGURE 1

Immobilized transcription complexes. (a) Leading-edge FRET. By labeling the leading edge of RNAP and the downstream end of DNA, we can monitor downstream translocation of RNAP by looking at increasing values of FRET. (b) Immobilization of the transcription complexes. Amino-silanized quartz slides are covalently modified by a layer of PEGs (1.25% biotinylated). The slides are incubated with streptavidin, rinsed, incubated again with 20–50 pM biotinylated transcription complexes, and rinsed before imaging. (c) DNA constructs: lacCONS (17) derivatives having no guanine residues on the template strand from +1 to +11. The doubly labeled DNA fragments are generated by PCR. (Boxes) Transcription start site (with arrow), promoter −10 element, and promoter −35 element; (shaded boxes) halt sites for RP_itc,≤2, RP_itc,≤4, RP_itc,≤7, and RD_e,11, respectively.

FIGURE 2

msALEX-DI, experimental setup; EOM, electrooptic modulator; DM, dichroic mirror. The image is split into two zones on the CCD camera, corresponding to the donor (left) and the acceptor (right) emission channels. The camera is synchronized with the alternation of the lasers, resulting in four images of the illuminated area (two excitations × two emissions).

FIGURE 3

Data analysis. (a) Representative raw data. After channels overlay and correction for optical aberrations, the molecules are identified (see Materials and Methods). The intensity corresponding to each molecule is integrated and background subtracted for each of the excitation/emission combinations, resulting in four intensities (), used to calculate the and S ratios. (b) Populations distributions. (Left) The and S ratios are displayed on a two-dimensional histogram. Donor-only (S > 0.8), acceptor-only (S < 0.2), and donor-acceptor complexes (0.2 < S < 0.8) are readily identified. (Right) Example of data obtained with a low- complex (RP_itc,≤2; Cy5, +25), displaying all the ( S) values at all time points, for all complexes. (c) Elimination of complications due to compositional heterogeneity. (Left) The acceptor-only population is removed by selecting the above a certain threshold (>1000 counts). (Right) The donor-only population is removed, by selecting the above a certain threshold (>1000 counts). As a result, only the ( S) values at all time points corresponding to D-A complexes are displayed. The relevant low peak is separated from the D-only peak (≈ 0), and its mean value can be accurately recovered from the projection of the histogram onto the axis. (d) Elimination of complications due to inactive state of the acceptor. (Left) Using single laser excitation (donor excitation), traces corresponding to donor ( green) and acceptor trough FRET ( red) emissions are obtained (top). The resulting trace, presented at the bottom, shows an interconversion between a high and a low state. (Right) msALEX-DI allows one to excite the acceptor directly and monitor its emitted fluorescence ( black). In this case, it is clearly shown that acceptor blinking (cycling between active and inactive states of the acceptor) is responsible for the observed anticorrelated behavior of the and traces. Removal of the time points where acceptor is inactive generates a new trace (bottom) where only time points with active acceptor are retained.

FIGURE 4

Promoter escape leading edge FRET (Cy5, +20); distributions, calculated by averaging values for each single complex, taking into account only the time points where donor and acceptor probes are active. Transition from initiation (top) to elongation (bottom) results in the appearance of a new population at ≈ 0.61. Inactive complexes are seen as an immobile population at ≈ 0.3. Translocation activity is 72%.

FIGURE 5

Abortive initiation (a–d). (Left) Schematic diagram depicting the heterogeneity and dynamic behavior of the RP_itc,≤2, RP_itc,≤4, and RP_itc,≤7 complexes engaged in abortive cycling (double-headed black arrows), as opposed to the static nature of the RD_e,11. (Center and right) histograms (for all time points where donor and acceptor probes are active) of D-A complexes for (a) RP_itc,≤2, (b) RP_itc,≤4, (c) RP_itc,≤7, and (d) RD_e,11, for LE-FRET experiments (Cy5, +20, and +25). Data were analyzed by fitting the RP_itc,≤2 histogram to a single Gaussian, followed by fitting the RP_itc,≤4 and RP_itc,≤7 histograms with a two-Gaussian function; in the second fit, one Gaussian function was constrained to have the mean and width values for RP_itc,≤2, and an amplitude equal to the fraction of complexes that fail to enter elongation (see text). The recovered values for the center and width of the distribution for the active molecules are: for Cy5, +20 (center (width)), RP_itc,≤2, 0.38 (0.45); RP_itc,≤4, 0.51 (0.43); RP_itc,≤7, 0.58 (0.45); and RD_e,11, 0.68 (0.31); for Cy5, +25 (center (width)), RP_itc,≤2, 0.11 (0.30); RP_itc,≤4, 0.24 (0.31); RP_itc,≤7, 0.29 (0.31); and RD_e,11, 0.51 (0.31). The vertical dotted line represents the mean value of the distribution for RP_itc,≤2. (e) Recovered values for the and D-A distances for the active molecules, as a function of the RNA product length (▵, Cy5, +20; ▪, Cy5, +25). For both constructs, transition from RP_itc,≤2 to RP_itc,≤4, to RP_itc,≤7 to RD_e,11 leads to an increase in the value (left), consistent with a decrease in distance between the leading edge of the enzyme and the downstream DNA (right).

FIGURE 6

Single-molecule time traces traces are plotted as a function of time, for different representative complexes (Cy5, +25). Only time points with active donors and acceptors are shown. The average values obtained for RP_itc,≤2 and RD_e,11 (0.11 and 0.5, respectively) are represented by two horizontal lines to guide the eye. (a) RP_itc,≤2, static distribution with ∼ 0.11 (see Fig. 5 a). (b) RP_itc,≤7, scattered distribution with a majority of time points distributed around a relatively high (t) value (distribution centered around = 0.3 for the active molecules (see Fig. 5 c)), consistent with the forward translocation of the leading edge of RNAP relative to downstream DNA during abortive initiation. (c) RD_e,11, static distribution with ∼ 0.5 (see Fig. 5 d).

FIGURE 7

Determination of correction factors for accurate FRET efficiency determination. (a) Determination of l, the leakage of the donor emission in the acceptor channel, and d, the acceptor emission due to the direct excitation of the acceptor by the donor-excitation laser; l is the center of the distribution for D-only molecules (top), and the d is the center of the distribution for A-only molecules (right). (b) Determination of γ, the factor that accounts for differences in detection efficiencies in the donor and acceptor emission channels. histogram for a RP_itc,≤2 (dark gray) and a RDe_,11 (light gray) sample. For each complex, (top) and (right) distributions are fitted with a Gaussian function to determine the center of the distribution. (c) Determination of ) values are plotted for different complexes, here RP_itc,≤2, RP_itc,≤7, and RD_e,11. The -factor is then calculated from the slope Σ and the intercept Ω of the best linear fit to the () values, as described in the Materials and Methods section.