Pleomorphic effects of three small-molecule inhibitors on transcription elongation by Mycobacterium tuberculosis RNA polymerase
- Jason L. Choy Laboratory of Single-Molecule Biophysics, University of California, Berkeley, United States
- Department of Molecular and Cell Biology, University of California, Berkeley, United States
- Laboratorio de Moléculas Individuales, Laboratorios de Investigación y Desarrollo, Facultad de Ciencias e Ingeniería, Universidad Peruana Cayetano Heredia, Peru
- California Institute for Quantitative Biosciences, University of California, Berkeley, United States
- Department of Chemistry, University of California, Berkeley, United States
- Department of Biomedical Engineering, School of Medicine, Shenzhen University, China
- Department of Chemistry & Biochemistry, University of Notre Dame, United States
- Waksman Institute, Rutgers University, United States
- Department of Chemistry and Chemical Biology, Rutgers University, United States
- Department of Physics, University of California, Berkeley, United States
- Biophysics Graduate Group, University of California, Berkeley, United States
- Kavli Energy Nanoscience Institute, University of California, Berkeley, United States
- Howard Hughes Medical Institute, University of California, Berkeley, United States
Figures
Figure 1
Single-molecule transcription elongation by M. tuberculosis RNA polymerase under high-resolution optical tweezers.
(a) (left) The experimental optical tweezer setup is shown, which tethers and isolates the RNA polymerase (RNAP) from the laser beams through a DNA handle and template. (right) A few single-molecule transcription traces are shown, with the pauses (red) and the pause-free periods (green) highlighted. (b) A diagram of the assisting constant force mode is shown. The traps are moved apart as the RNAP transcribes to keep the force constant. (c) A diagram of the opposing constant force mode is shown. The traps are moved closer as the RNAP transcribes to keep the force constant. (d) (left) Representative trajectories of transcription by Escherichia coli RNA polymerase (EcoRNAP) (black) and Mycobacterium tuberculosis RNA polymerase (MtbRNAP) (blue) under ~18 pN assisting constant force and saturating concentration of rNTPs (~1 mM) are shown. The DNA template for MtbRNAP derives from M. tuberculosis genes rpoB and rpoC. (right) Velocity distributions of these trajectories are shown, with a fit to a sum of two Gaussians, one with mean zero (pauses) and one with positive mean (pause-free velocity). (e) The populations and rates obtained via dwell time distribution (DTD) analysis are shown for EcoRNAP and MtbRNAP on Eco and Mtb templates. Error bars are 95% CIs of fits of steps obtained from (left to right) 16, 19, 24, and 16 molecules. The traces were collected under saturating concentrations of nucleotides and 15–20 pN of assisting constant force.
Figure 2
Mechanochemical characterization of the elongation by M. tuberculosis RNA polymerase under high-resolution optical tweezers.
(a) The effect of magnitude and direction of applied force on the kinetic parameters obtained from dwell time distribution (DTD) analysis is shown. Positive forces are assisting constant forces, negative are opposing constant forces. Error bars are 95% CIs of fits of steps from (left to right) 4, 6, 10, 17, 14, 9, 3, 4, and 5 molecules. (b) A diagram of opposing passive mode is shown. The traps are held stationary as the RNA polymerase (RNAP) transcribes, leading to an increase in force. (c) Example opposing passive mode traces are shown. The Mycobacterium tuberculosis RNA polymerase (MtbRNAP) transcribes at near constant velocity before suddenly stalling and backtracking. (d) Force-velocity relationships for MtbRNAP and Escherichia coli RNA polymerase (EcoRNAP) obtained from passive mode experiments are shown and fit to a generalized Boltzmann relation (right). Both polymerases' velocities remain insensitive to force until they reach similar stall forces, on average 19.3 pN ± 1.2 pN for MtbRNAP, and 17.1 ± 1.1 pN for EcoRNAP. Error bars are standard deviations and come from 14 (Eco) and 22 (Mtb) molecules.
Figure 3 with 1 supplement
Mycobacterium tuberculosis RNA polymerase (MtbRNAP) pauses less efficiently than Escherichia coli RNA polymerase (EcoRNAP) on Eco pauses.
(a) Representation of the template containing a molecular ruler to study EcoRNAP pauses. The DNA template consists of eight repeats of five Eco elementary pauses, a, b, c, d, and his. (b) Example traces of single Eco and Mtb RNA polymerases transcribing the E. coli molecular ruler are shown. The eight repeats are denoted as vertical arrows, and the location of the pauses within each repeat are shown as horizontal colored lines. We collected the transcription activities using a cocktail of rNTPs (1 mM rUTP, 1 mM rGTP, 0.5 mM rATP, and 0.25 mM rCTP) at 15–20 pN for EcoRNAP and 18 pN for MtbRNAP. The zoom on the right shows the alignment of the trace pauses with the expected pause locations. (c) Average residence time histograms of EcoRNAP and MtbRNAP display distinctive patterns of pause strength (peak height) and location. MtbRNAP more weakly recognizes the Eco pauses. (d) Comparison of the transcription gel bands at the elemental pause sites between MtbRNAP and EcoRNAP.
-
Figure 3—source data 1
Original raw gels for Figure 3d.
- https://cdn.elifesciences.org/articles/105545/elife-105545-fig3-data1-v3.zip
-
Figure 3—source data 2
Full gel images for Figure 3d, with labels.
- https://cdn.elifesciences.org/articles/105545/elife-105545-fig3-data2-v3.zip
Figure 3—figure supplement 1
The design of a molecular ruler to study pausing in Mycobacterium tuberculosis RNA polymerase (MtbRNAP) with high-resolution optical tweezers.
(a) An artificial transcription bubble scheme with a 5'-FAM-labeled RNA was used for a bulk elongation assay. The stalled ternary elongation complex at position G15 (TECG15) resumes RNA polymerization activity upon adding 250 µM rNTPs, and pauses at pause c (TECA26). (b) MtbRNAP efficiently recognizes sequence derived from E. coli elemental pause c. (left) A urea PAGE gel that shows the clearance of the pause site at TEGA26 with a mean duration of ~ 27 s (inverse of the pause escape rate). (right) The quantification of the bands and their fitting to a bi-exponential decay. The extracted parameters indicate that the pause escape rate is similar in duration to the his pause of Escherichia coli RNA polymerase (EcoRNAP) (Larson et al., 2014). (c) The Mtb molecular ruler enables the analysis of sequence-dependent pausing by MtbRNAP at the single-molecule level. Example traces are shown on the left, collected at 1 mM rNTPs and 18 pN assisting constant force. Below is a zoom around the designed pause. The residence time histogram (RTH) on the right shows the strong pausing at 50 bp, and how the strength changes with the direction of the applied force (18 pN assisting constant force vs. 9 pN opposing constant force).
-
Figure 3—figure supplement 1—source data 1
Original raw gels for Figure 3—figure supplement 1b.
- https://cdn.elifesciences.org/articles/105545/elife-105545-fig3-figsupp1-data1-v3.zip
-
Figure 3—figure supplement 1—source data 2
Full gel images for Figure 3—figure supplement 1b, with labels.
- https://cdn.elifesciences.org/articles/105545/elife-105545-fig3-figsupp1-data2-v3.zip
Figure 4 with 4 supplements
The inhibition of Mycobacterium tuberculosis RNA polymerase (MtbRNAP) by D-IX216 involves the conversion to one of two slowly elongating inhibited states.
(a) Example traces of MtbRNAP transcribing in the absence (gray) and presence (red) of 140 nM D-IX216 are shown. (b) Traces exhibiting the three states, fast, slow, and super-slow, are shown and the regions are colored blue, orange, and red, respectively. Transcription in the absence of D-IX216 is shown in gray. The traces plotted in this figure were collected at 18 pN of assisting constant force, 1 mM rNTPs, and 140 nM D-IX216. (c) Comparison of the complementary cumulative distribution function (CCDF) of the dwell time distributions (DTD) of the fast, slow, and super-slow states. (d) DTD analysis of the fast, slow, and super-slow inhibited states are shown. Pf: pause-free, ep: elemental pause, lp: long pause. Error bars are 95% CIs of fits of steps obtained from 60 molecules. (e) (left) MtbRNAP traces crossing the Mtb molecular ruler are shown in varying states of inhibition. (right) The residence time histograms of these inhibited states are shown both in seconds or normalized to the median residence time.
Figure 4—figure supplement 1
D-IX216 specifically slows down the Mycobacterium tuberculosis RNA polymerase (MtbRNAP) transcription.
(a) The chemical representations of the Nα-aroyl-N-aryl-phenylalaninamides D-AAP1 and D-IX216 are shown. Rings A and C are modified to increase the affinity of D-IX216 for MtbRNAP (Ebright et al., 2015). (b) D-IX216 does not alter Escherichia coli RNA polymerase (EcoRNAP) activity. An example trace of a two-shunt experiment shows that 280 nM D-IX216 does not affect EcoRNAP activity. The dotted boxes denote what solution is flowing out of the shunt at what time. (c) The kinetic parameters obtained from dwell time distribution (DTD) analysis of EcoRNAP in the absence and presence of D-IX216 is shown. In this dataset, there were too few events to fit a third exponential (long pauses). Error bars are 95% CIs of fits of steps from 19 (no D-IX216) and 6 (with D-IX216) molecules. (d) This buffer exchange control experiment shows that the RNA polymerase (RNAP) quickly responds to buffer changes. When nucleotides are removed, MtbRNAP immediately pauses until the nucleotides are reintroduced. (e) D-IX216 slows down MtbRNAP rather than pausing or halting it. The trajectory of a single MtbRNAP elongating at constant 18 pN assisting constant force with 280 nM D-IX216 and saturating nucleotides (~1 mM) shows a slow state that eventually instantly recovers its velocity (~ 20 bp/s). Depending on the condition, up to ~41% of polymerases can recover their initial velocity. The dotted boxes show the start and duration of the shunt opening to supplement the inhibitor and nucleotides. The apparent association time of D-IX216 binding corresponds to the time between the shunt opening to introduce D-IX216 and the switch’s appearance. On the other hand, the apparent D-IX216 dissociation time corresponds to the slow region’s lifetime. (f) D-IX216 is still engaged during MtbRNAP elongation. We performed a buffer exchange using two shunts to remove the inhibitor from the affected polymerase. However, this did not immediately restore its global velocity. Towards the end of the trace, the polymerase might detach from the DNA template, leading to tether break events or stalls in 45% and 13% of the polymerases, respectively.
Figure 4—figure supplement 2
Interconversion between the slow and super-slow inhibited states.
(a) The complementary cumulative distribution functions (CCDFs) of lifetimes of the slow and super-slow inhibited states are shown with a fitting to a single exponential decay. The lifetime was measured as the time between switching into that state, until the motor either recovered (fast) or switched into the other state (e.g. a switch from slow to super-slow). Tether-break events were not included. (b) Comparison of the processivity in terms of kb extended by the elongating polymerase before tether break (template dissociation), or in terms of kb before a switch event of the super-slow and slow inhibited states is shown as a quartile plot from 60 molecules. Outliers in this data were taken as points farther than 1.5*IQR from the upper or lower quartile, and are shown as + marks. Processivity is lower in the super-slow inhibited state than in the slow inhibited state by both definitions. The data for the bar plots were obtained from combined data of assisting and opposing constant force experiments. (c) Transition maps under assisting and opposing constant force of the different states (fast, slow, and super-slow) are shown.
Figure 4—figure supplement 3
The fast elongating state of Mycobacterium tuberculosis RNA polymerase (MtbRNAP) observed in the presence of D-IX216 corresponds to the inhibitor-free state.
(a) Example fittings (colored) of the dwell times to the raw data (gray) for the inhibitor-free and the various inhibited states of MtbRNAP are shown. (b) The complementary cumulative distribution functions (CCDFs) of the dwell time distributions (DTDs) for fast elongating polymerases observed with (black and dark blue) and without D-IX216 (cyan). (c) The kinetic parameters from DTD analysis for the enzyme without D-IX216 and the enzyme in the fast state with D-IX216 are shown. No notable differences are found between the inhibitor-free and the fast state with D-IX216. Error bars are 95% CIs of fits of steps from 19 (no D-IX216) and 60 (with D-IX216) molecules.
Figure 4—figure supplement 4
Effect of different D-IX216 and nucleotide concentrations on the slow and super-slow inhibited states of Mycobacterium tuberculosis RNA polymerase (MtbRNAP).
(a) The concentration of D-IX216 did not impact the kinetic parameters obtained by dwell time distribution (DTD) analysis of MtbRNAP in the slow inhibited states. This suggests that a single molecule of D-IX216 causes the conversion into the slow state. Error bars are 95% CIs of fits of steps from (left to right) 3, 60, 9, 10, and 12 molecules. (b) Measurement of the dissociation constant (KD) of D-IX216 for the elongating MtbRNAP. The association and dissociation times obtained from the duration of the fast, slow, and super-slow inhibited states in the single-molecule traces are fit to single-exponentials to obtain kon and koff. Their ratio is KD. (c) The concentration of rNTPs did not affect the kinetic parameters in the slow inhibited state of MtbRNAP induced by D-IX216. This indicates that the slowing by D-IX216 is not due to a reduction in nucleotide affinity. These experiments were done in semipassive assisting force mode, where the optical traps are moved stepwise to keep the force within a set range. In other words, the traps are held at constant position, causing the force to fall as transcription progresses, until it reaches some lower force limit (in this case, 10 pN) after which the traps move apart to raise the force to some upper force limit (in this case, 18 pN). Error bars are 95% CIs of fits of steps from (left to right) 6, 2, 8, 21, 11, and 17 molecules.
Figure 5 with 1 supplement
Streptolydigin inhibits Mycobacterium tuberculosis RNA polymerase (MtbRNAP) by enhancing pausing and inducing backtracking during pausing.
(a) Example traces of MtbRNAP transcription in the absence (gray) and presence (green) of 15 µM Stl, with 18 pN of assisting constant force and saturating rNTP concentrations. (b) Details of a backtracking event induced by Stl are shown, exhibiting a process where there is both an initial pre-backtrack pause, a backtrack, and then recovery from that backtrack. (c) Fittings for the complementary cumulative distribution functions (CCDFs) of the pre-backtrack and recovery times of Stl-induced pauses to single exponentials are shown. (d) The comparison between the fitting of the backtrack recovery between two models is shown: a single-exponential distribution corresponding to deep backtracking and a power law distribution corresponding to shallow backtracking. (e) The kinetic parameters obtained from dwell time distribution (DTD) analysis in the presence of Stl are shown, under a constant 18 pN assisting or 4 pN opposing constant forces. Error bars are 95% CIs of fits of steps obtained from (left to right) 19, 22, 14, and 17 molecules. (f) Average residence time histograms of MtbRNAP transcribing the repeat of the molecular ruler in the absence and presence of Stl is shown. (g) Kinetic parameters extracted from DTD analysis of MtbRNAP traces under constant 5 pN of opposing force and 15 µM Stl in the absence or presence of 0.5 µM MtbGreA are shown. Error bars are 95% CIs of fits of steps obtained from 17 (-MtbGreA) and 9 (+MtbGreA) molecules.
Figure 5—figure supplement 1
Streptolydigin (Stl) induces reduced post-translocated states in Mycobacterium tuberculosis RNA polymerase (MtbRNAP).
(a) A bulk assay was performed to measure the translocation of RNA polymerase (RNAP) by one nucleotide using the Mtb ternary elongation complex. The technique involved real-time measurement of the fluorescent enhancement at 375 nm of a nitrogenous base analog, 2-aminopurine52. This analog was positioned on the template strand of the DNA-RNA hybrid, initially at position +2, and then moved towards position +1 due to the RNAP translocation process. The workflow included the use of a fluorescent artificial bubble to measure the effect of Stl on the translocation state of MtbRNAP. After the RNAP incorporated one rATP, the translocation state was measured by the dye’s fluorescence in the following base in the template DNA. When the base was paired, fluorescence was low due to quenching; when the base was unpaired, such as in the post-translocated state, the fluorescence was high. The assay conditions consisted of transcription buffer and 100 µM rATP, along with either 100 µM dATP, 0.25 µM MtbGreA, 7.5 µM Stl, or 0.25 µM MtbGreA / 7.5 µM Stl. (b) The comparison of different conditions of the translocation trace of the MtbRNAP in the presence of cognate nucleotide (rATP) and Stl shows that the inhibitor allows only ~20% of the MtbTEC to attain the post-translocated state (green dots). The addition of MtbGreA was used as a positive control for post-translocation.
Figure 6
Pseudouridimycin (PUM) inhibits Escherichia coli RNA polymerase (EcoRNAP) by enhancing pausing and infrequently inducing slowly elongating inhibited states.
(a) Single-molecule traces of EcoRNAP transcription in the absence (black) and presence (purple) of 1 µM PUM are shown. Traces were collected at 10 pN constant assisting force and 1 mM rNTPs. (b) A comparison of the fast state and the infrequent slow and super-slow inhibited states with PUM are shown. (c) Kinetics parameters attained from dwell time distribution (DTD) analysis of the three regions of EcoRNAP with PUM are shown. Pf: pause-free, ep: elemental pause, lp: long pause. Error bars are 95% CIs of fits of steps obtained from 27 molecules. (d) (left) Example traces of EcoRNAP crossing the molecular ruler in the presence of PUM are shown. (right) The average residence time histogram of EcoRNAP crossing one repeat is shown at varying ratios of PUM to 1 mM rUTP.
Figure 7 with 1 supplement
Inhibition of Mycobacterium tuberculosis RNA polymerase (MtbRNAP) by pseudouridimycin (PUM) involves induction of slowly elongating inhibited states.
(a) (left) Single-molecule MtbRNAP transcription in the absence (gray) and presence (purple) of 1 μM PUM are shown. (right) Examples of transcription in the slow inhibited state, the super-slow inhibited state, and a dynamic pause are shown. Transcription was carried out under 18 pN of assisting constant force and 1 mM rNTPs. (b) Kinetic parameters attained from dwell time distribution (DTD) analysis of the three regions of MtbRNAP with PUM are shown. Pf: pause-free, ep: elemental pause, lp: long pause. Error bars are 95% CIs of fits of steps obtained from 27 molecules. (c) The average residence time histogram of a repeat of MtbRNAP transcribing the Mtb molecular ruler in the presence and absence of 1 μM PUM is shown.
Figure 7—figure supplement 1
Comparison of events found in transcription with antibiotics.
(a) Example traces in the slow inhibited state caused by pseudouridimycin (PUM) and D-IX216 are shown. (b) Example traces in the super-slow inhibited state caused by PUM and D-IX216 are shown. (c) Comparison of a common pause obtained in the absence of inhibitor versus a dynamic pause caused by PUM is shown. (d) Comparison of a backtrack event observed in the presence of streptolydigin (Stl) compared to a rare non-traditional backtrack event induced by PUM is shown.
Figure 8
Inhibition of Mycobacterium tuberculosis RNA polymerase (MtbRNAP) by a combination of D-IX216 and streptolydigin (Stl).
(a) Comparison of transcription elongation traces of single molecules of MtbRNAP obtained with D-IX216, Stl, or both are shown. (b) A zoom of the traces with D-IX216 and Stl just before arrest are shown. The blue dotted guide lines match the average velocity of MtbRNAP in the fast, slow, and super-slow inhibited states from left to right.
Additional files
-
Supplementary file 1
Tables of oligos, plasmids and cells.
- https://cdn.elifesciences.org/articles/105545/elife-105545-supp1-v3.docx
-
Supplementary file 2
Summary of traces.
- https://cdn.elifesciences.org/articles/105545/elife-105545-supp2-v3.xlsx
-
MDAR checklist
- https://cdn.elifesciences.org/articles/105545/elife-105545-mdarchecklist1-v3.docx
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Pleomorphic effects of three small-molecule inhibitors on transcription elongation by Mycobacterium tuberculosis RNA polymerase
eLife 14:e105545.
https://doi.org/10.7554/eLife.105545
