Single-molecule study of transcriptional pausing and arrest by E. coli RNA polymerase

Science

Volumn 287, Issue 5462, 2000, Pages 2497-2500

  Davenport, R John ^a   Wuite, Gijs J L ^a   Landick, Robert ^b   Bustamante, Carlos ^a,c  

^a UNIVERSITY OF CALIFORNIA   (United States)

^b UNIVERSITY OF WISCONSIN MADISON   (United States)

^c LAWRENCE BERKELEY NATIONAL LABORATORY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

RNA POLYMERASE;

ARTICLE; DNA TEMPLATE; ENZYME CONFORMATION; ENZYME KINETICS; ENZYME STABILITY; ESCHERICHIA COLI; GENE CONTROL; GENETIC ANALYSIS; MICROSCOPY; NONHUMAN; PRIORITY JOURNAL; RNA TRANSCRIPTION; TRANSCRIPTION REGULATION;

DNA, BACTERIAL; DNA-DIRECTED RNA POLYMERASES; ESCHERICHIA COLI; KINETICS; MICROSCOPY, VIDEO; MODELS, GENETIC; OPTICS; RNA, BACTERIAL; RNA, MESSENGER; TEMPLATES, GENETIC; TRANSCRIPTION, GENETIC;

ESCHERICHIA COLI; EUKARYOTA; NEGIBACTERIA;

EID: 0034737593     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.287.5462.2497     Document Type: Article

References (35)

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17. A water immersion, high numerical aperture (NA = 1.2) objective lens with a 220-μm working distance allows the stable trapping of a micrometer-sized sphere 100 μm from the cover slip surface of the fluid chamber with a single-laser beam optical trap (λ = 835 nm). This design makes it possible to introduce a micropipette in the chamber to hold a bead by suction. The bead in the trap and the bead on the pipette function as easily exchangeable surfaces between which RNAP molecules can be attached. A computer-controlled flow system is used to exchange the liquid in the fluid chamber and to establish a flow rate with high precision. The flow can be used to exert force on the beads, whose displacements are then measured by video microscopy (at 10 Hz in real time). The instrument is highly automated to maximize experimental throughput. Individual stalled elongation complexes were assembled between two beads, one held in a laser trap and the other held on a micropipette. After assembly, the trapped bead was released from the trap to avoid laser damage to the transcription complex. The complex was then extended by drag applied on the bead. The flow could be increased or decreased to vary the hydrodynamic drag force on the tethered bead, and therefore, the load force acting on the polymerase. Upon addition of NTPs, we followed the resumption of transcription by the shortening of the DNA tether between the beads. A movie of transcription by a single molecule of RNAP can be viewed at http://alice.berkeley.edu/RNAP
18. The position resolution of a tethered bead is ∼21 base pairs (bp) (∼7 nm). This position resolution is determined by the rms displacement due to brownian motion after averaging over 0.033 s (∼4 nm) added to the error of the bead centroid determination (∼3 nm) (34). The tethered particle motion method has a resolution of 400 bp using a DNA fragment 10 times shorter and averaging over 0.27 s (15).
19. B is the Boltzmann constant, and T is the absolute temperature.
20. corr introduces an alignment error of the different data sets resulting in ∼20 nm total error.
21. 2. Brownian motion limits the resolution of rate determination to ± 1 bp/s (2 SD) as determined from the distribution of rate fluctuation of beads tethered to nontranscribing polymerases. The quantitative criterion used to score whether an enzyme has paused is the following. A pause is scored if the rate of transcription at that location is below the limit of rate resolution (1 bp/s). The rate at the local rate minimum was determined by fitting a line to the position versus time data for the duration of the pause (26). This analysis made it possible to distinguish between pauses and locations in which the enzyme had simply slowed down but had not paused.
22. Analysis of the enzyme population displaying this bimodal distribution reveals that ∼75% of the molecules exist in a slow state for most of the time that they transcribe (these are often RNAP enzymes which transcribed for shorter times). The fast-transcribing enzymes, on the other hand, were more prone to change rate to become slower transcribers. The different heights of the peaks in the bimodal distribution are likely to arise from this difference in switch behavior between the two populations. Most RNAP enzymes that transcribe longer distances (and times) undergo one, or sometimes more, rate changes. Changes in peak rate up to 18 bp/s have been observed for some molecules. The most frequent changes in peak rate are ±2bp/s (n = 18), followed by changes of ±4 to 5 bp/s (n = 11). Changes smaller than ±1 bp/s are indistinguishable from Brownian motion (21). The most frequent rate changes are sufficient to allow a molecule to jump from the slow to the fast transcribing state in the histogram in Fig. 3A, and vice versa.
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26. The pause duration is taken as the time it takes an enzyme to move from a position 30 bp before the pause to another location 30 bp after the pause (twice the spatial resolution).
27. The precise template position of a pause cannot be determined at the present resolution of the experiment (20, 34). However, the average spacing between detected pauses (∼200 bp) is ∼3.5 times the spatial uncertainty in their position. Some of the pausing events detected may, nonetheless, correspond to several close-lying pauses. On average, molecules translocate for about 75 s before pausing. This figure is five times longer than the filter frequency used on the data. Thus, the majority of pauses are detected.
28. The same nine peaks were present when similar histograms were generated from 23 data sets picked randomly from the complete data set.
29. If pausing corresponds to a state on the same kinetic pathway as normal elongation, then the half-life and "apparent efficiency" at each pause site would be correlated to one another, since both depend on the forward rate constant for the next template position. In other words, if a site had a relatively long half-life, this long residence time would make it more likely that the pause will be scored, thus introducing a correlation between the half-life and the apparent efficiency.
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34. The bead position is determined by averaging the positions of all the pixels inside the circular black edge of the bead image (the resolution of this procedure is ∼3 nm). The experimental uncertainty in the determination of the center-to-center bead distance for a 2.2-μm diameter bead held at the end of a 3.5-μm-long DNA molecule extended by a drag force of 5.5 pN is 9.8 nm (i.e., ∼4 nm of rms displacement due to Brownian motion plus twice the error in the bead centroid determination, or ∼6 nm).
35. Supported by NIH grant GM-32543 and NSF grants MBC 9118482 and DBI 9732140 to C.B. and by NIH grant GM-38660 to R.L. The authors thank C. Rivetti, D. Erie, and F. Stahl for providing tools for the construction of the DNA template and S. B. Smith, M. Hegner, Z. Bryant, and M. Young for many helpful discussions.