Potent transcriptional interference by pausing of RNA polymerases over a downstream promoter - PubMed (original) (raw)

Potent transcriptional interference by pausing of RNA polymerases over a downstream promoter

Adam C Palmer et al. Mol Cell. 2009.

Abstract

Elongating RNA polymerases (RNAPs) can interfere with transcription from downstream promoters by inhibiting DNA binding by RNAP and activators. However, combining quantitative measurement with mathematical modeling, we show that simple RNAP elongation cannot produce the strong asymmetric interference observed between a natural face-to-face promoter pair in bacteriophage lambda. Pausing of elongating polymerases over the RNAP-binding site of the downstream promoter is demonstrated in vivo and is shown by modeling to account for the increased interference. The model successfully predicts the effects on interference of treatments increasing or reducing pausing. Gene regulation by pausing-enhanced occlusion provides a general and potentially widespread mechanism by which even weak converging or tandem transcription, either coding or noncoding, can bring about strong in cis repression.

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Figures

Figure 1

Figure 1. Activation of PRE by IPTG induction of CII

A) Diagram of single-copy chromosomal lacZ reporter and plasmid system for IPTG regulated expression of CII. IPTG regulated expression of CII is achieved with a two plasmid system: pUHA1 contains pLacI-lacI and pZS15_cII_ contains pLac-cII. The region of lambda used in the reporter system is shown as a thicker line. The relative locations of the Rho utilisation site (rut), N utilisation site (nut) and the tR1 terminator are shown. (B) Activity of PRE.(PR−).lacZ (n=12) as a function of IPTG, which induces expression of CII from pLac in pZS15_cII_. Error bars in this and all subsequent figures are 95% confidence limits. The line connecting points is the Hill function of best fit. The average Hill coefficient is 5.1±0.4 (95% confidence limits). (C) Western blotting of CII from E. coli pUHA1 pZS15_cII_ grown in a range of IPTG concentrations, quantitated against a calibration curve of pure CII protein added to _cII_− E. coli extracts. For the IPTG concentrations 100 to 300μM (lower panel), cII+ extracts were diluted 3.3-fold into _cII_− extracts, to give a quantity of CII that lies within the calibration curve. The 100μM IPTG point was measured in both diluted and undiluted form, giving average measurements within 6% of one another. (D) Quantitation of CII western blotting, showing the results of three independent sets of western blots (filled, open, and grey). Grey circles are the data of (C), and a continuous line shows the average of all data. (E) Activity of PRE.(PR−).lacZ plotted as a function of CII molecules per cell. Using the western blotting of (C) and (D), CII molecules per cell were measured for 7 IPTG concentrations. CII molecules per cell for those IPTG concentrations not directly measured were determined by interpolation between measured data. The average Hill coefficient is 3.1±0.3 (95% confidence limits). Assuming an average cellular volume of 1.4 fL, we can determine from this data that the in vivo affinity of CII for its binding site is KD = 3.7 μM.

Figure 2

Figure 2. Transcriptional interference between PR and PRE

(A) Activity of PRE.(PR−).lacZ and PRE.(PR+).lacZ (n=14), demonstrating a 5.5-fold repression of PRE due to TI from PR. Lines are Hill functions of best fit; the fitted EC50s are 78μM IPTG for PRE.(PR−) and 76μM IPTG for PRE.(PR+). (B) Activity of PR (short).lacZ (n=6), PR.(PRE+).lacZ (n=15), and pBla.lacZ (n=10), in response to IPTG induction of CII. Only PR.(PRE+).lacZ contains a CII binding site or CII-activated promoter in between the promoter and lacZ.

Figure 3

Figure 3. Simulations of transcriptional interference cannot explain experimental observations using existing mechanisms

(A) Schematic of different mechanisms of transcriptional interference, in which the left (darker) promoter is interfering with the right (lighter) promoter. (B) Alignment of experimental measurements of promoter activity with the existing mathematical model of TI. Points are experimental data and lines are the results of stochastic simulations of TI, incorporating the mechanisms of occlusion, ‘sitting duck’, and collisions.

Figure 4

Figure 4. RNAP from pR pauses at tR1

(A) Sequence of tR1 and PRE. Above the sequence the three in vitro pause/termination sites at tR1 (Lau et al., 1982) are marked, together with the expected protected region for RNAP paused at each of these pause sites. Below the sequence is marked the binding region for RNAP at PRE, demonstrating that RNAP paused at tR1 should sterically hinder the association of RNAP to PRE. (B) In vivo potassium permanganate footprinting was performed to identify RNAP pause sites at tR1, for _rut_A+ and _rut_A− templates. To ensure pauses were specific for transcription originating from pR, pR+ and pR− templates were compared. Footprints were obtained for both top and bottom strands, and run alongside lanes containing dideoxy sequencing reactions which had been generated using the same primers (only the A lane is shown). The bands which are the most distinctly pR specific are indicated by arrowheads. The indicated bands were observed in several (n=5) independent footprint reactions and are consistent with the known three tR1 termination sites observed in vitro (indicated at the side of figure).

Figure 5

Figure 5. A _rut_A mutation extends the lifetime of the pauses

(A) Measurement of pause durations at the three tR1 sites by in vivo permanganate footprinting following addition of rifampicin. Footprints were obtained for the bottom strand on both _rut_A+ and _rut_A− templates and run alongside lanes containing dideoxy sequencing reactions which had been generated using the same primer (only the A and C lanes are shown). The appearance of a strong pR band indicates accumulation of open complexes at pR, showing that rifampicin is blocking further rounds of RNA synthesis. In contrast, the tR1 pause signals decay with time, as the paused RNAP either terminates or resumes elongation. Plots of pixel intensity down the _rut_A+ lanes of the gel are overlaid for the 15 (blue), 30 (green), 60 (red) and 300 (black) second time points. Black dots indicate the pR-specific bands (Figure 4B) which were used for the estimation of pause durations at sites I, II and III shown in (B). (B) The average RNAP pause durations for _rut_A+ and _rut_A− templates at each of the three tR1 pause sites were estimated by plotting the rate of loss of signal with time following addition of rifampicin. The average pause durations, calculated as 1/slope of these plots, are indicated within each graph. The intensity of the 15 second time point was used as the initial value, in order to allow time for pR derived polymerases, which were elongating at the time of rifampicin addition, to reach tR1. Average pause durations were consistently increased on the _rut_A− templates.

Figure 6

Figure 6. Occlusion by paused RNAP can explain the strong transcriptional interference of PRE

(A) Experimental and simulated TI of PRE, in _rut_A+ and _rut_A− conditions, with simulations incorporating occlusion by paused RNAP. Points are experimental data: PRE.(PR−).lacZ (blue), PRE.(PR+).lacZ (red), and PRE.(_rut_A− PR+).lacZ (black) (n=7). PRE.(_rut_A− PR+).lacZ has been scaled up to normalise PRE activity in the absence of TI, to facilitate comparison of fold-interference against _rut_A+ constructs; this is necessitated by _rut_A− causing a 27% decrease in PRE LacZ activity in the absence of TI. Lines are stochastic simulations of TI: dotted lines are simulations without RNAP pausing at tR1, and solid lines are simulations with intrinsic RNAP pause durations at tR1 of 20 seconds. Marked along the right of the graph are the predicted maximum activities of PRE.(PR−).lacZ (red) and PRE.(_rut_A− PR+).lacZ (black) with intrinsic pause durations of 0, 2, 5 and 20+ seconds, illustrating how pausing of RNAP progressively increases repression of PRE. The thickness of the 20+ second line spans the range of promoter activity calculated for pause durations from 20 to 1000 seconds. (B) As for (A), showing the effects of reduced pausing due to λN in green: PRE.(PR+ N+).lacZ (n=10). Expression of λN had no influence upon PRE.(PR−).lacZ (data not shown).

Figure 7

Figure 7. Substantial occlusion can only be obtained with RNAP pausing

(A) Analytical model of occlusion by RNAP pausing. RNAP with an occlusion length of l bp, travelling at velocity v bp/sec, arrive at an operator of length m bp, with flux f (RNAP/second). The RNAP move to the end of the operator with rate v.(l + m) −1, whereupon they reach a pause site and remain paused for an average duration P sec. Thus with rate _P_−1 the paused state can change to a vacant state, or with rate f another RNAP may arrive at the start of the operator. The fraction of time the operator is occluded is given by the equation shown. This result is independent of whether the pause site is positioned at the start or end of the operator. (B) Occlusion as a function of pause duration and RNAP flux, calculated for a m = 10bp operator being occluded by l = 30bp long RNAPs which elongate at a rate of v = 60 bp/sec. (C) Genetic arrangements where occlusion by pausing may regulate gene expression. Transcription may be tandem (upper) or convergent (lower) to the interfered promoter and may be either coding or noncoding. Pausing of RNAP over the promoter itself, or over associated elements such as transcription factor binding sites (TF) or enhancer sequences, may lead to reduction in promoter activity.

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