The elemental mechanism of transcriptional pausing.

Reagent type (species) or resource Designation Source or reference Identifiers Additional information Strain, strain background ( E. coli ) BL21λDE3 Novagene-EMD Millipore expression strain Recombinant DNA reagent 6-MI-containing oligonucleotides Fidelity Systems see Supplementary file 1 for sequences Recombinant DNA reagent DNA and RNA oligos Intergrated DNA Technologies (IDT) see Supplementary file 1 for sequences Chemical compound, drug Cystamine dihydrochloride MP Biomedicals Cat# ICN10049205 Chemical compound, drug Dithiothreitol (DTT) Gold Biotechnology Cat# DTT100 Chemical compound, drug Iodoacetamide Sigma-Aldrich Cat# I6125 Chemical compound, drug Heparin, Na salt Sigma-Aldrich Cat# H-3393 Chemical compound, drug Ribonucleotide triphosphates Promega Cat# P1221 grade I-A (181 USP units/mg) Chemical compound, drug Guanosine triphosphate (GTP) GE Healthcare Cat# 27-2076-01 Low background flouresence Chemical compound, drug [γ- 32 P]ATP PerkinElmer Life Sciences Cat# BLU002Z Chemical compound, drug [α- 32 P]CTP PerkinElmer Life Sciences Cat# BLU006H Chemical compound, drug [α- 32 P]GTP PerkinElmer Life Sciences Cat# BLU006H Chemical compound, drug Acrylamide-Bisacrylamide Bio-Rad Cat# 1610145 (19:1, 40% solution) Software, algorithm ImageJ NIH ( https://imagej.nih.gov/ij/ ) Software, algorithm Kaleidagraph Synergy Software Software, algorithm Excel Microsoft Software, algorithm ImageQuant GE Healthcare Software, algorithm KinTek Explorer v6.1 KinTek Corp. Other Phast gels GE Healthcare Cat# 17–067801 Other HiTrap Heparin HP column GE Healthcare Cat# 17-0406-01 Other HisTrap HP column GE Healthcare Cat# 17-5247-01 Other Streptactin 5 ml High Capacity Column IBA Cat# 2-1238-001 Reagents and materials Plasmids and oligonucleotides are listed in Supplementary file 1 . RNA and DNA oligonucleotides were obtained from Integrated DNA Technologies (IDT; Coralville, IA) and purified by denaturing polyacrylamide gel electrophoresis (PAGE) before use. GreA, GreB, and RNAPs were purified as described previously ( Larson et al., 2014 ; Windgassen et al., 2014 ). Briefly, His-tagged RNAPs were overexpressed in E. coli BL21 λDE3 and cells were lysed by sonication. RNAPs were enriched by PEI and ammonium sulfate precipitation, then purified by sequential nickel (5 mL HisTrap) and heparin (5 mL HiTrap) column chromatography, dialyzed into storage buffer (20 mM Tris-Cl, pH 8, 250 mM NaCl, 20 μM ZnCl 2 , 1 mM MgCl 2 , 0.1 mM EDTA, 1 mM DTT, and 25% glycerol), and stored in small aliquots at –80° C. In vitro transcription pause assays PAGE-purified 15-mer RNA with 3′ end two nt upstream from the pause site (5 µM) and template DNA (10 µM) were annealed in transcription buffer 1 (TB1; 20 mM Tris-OAc pH 7.7, 5 mM Mg(OAc) 2 , 40 mM KOAc, 1 mM DTT; sequences of RNAs and DNAs are in Supplementary file 1 ). Scaffolds were incubated with RNAP for 15 min at 37°C in TB1, then non-template DNA was added and incubation continued for 15 min at 37°C. The ratio of RNA:tDNA:RNAP:ntDNA was 1:2:3:5 (0.5 µM, 1 µM, 1.5 µM, 2.5 µM, respectively). ECs were diluted to 0.1 µM with TB1 + heparin (0.1 mg/ml), incubated for 3 min at 37°C, labeled by the incorporation of [α- 32 P]GMP at 10 µM total GTP for 1 min at 37°C, and then placed on ice for 30–60 min. ECs were incubated for 3 min at 37°C before initiating the pause assay by addition of CTP to 100 µM and GTP to 10 or 100 µM in TB1 at 37°C. Reaction samples were removed at various time points and quenched with an equal volume of 2X stop buffer (8 M urea, 50 mM EDTA, 90 mM Tris-borate buffer, pH 8.3, 0.02% each bromophenol blue and xylene cyanol). All remaining active ECs were chased to product by incubation with GTP at 1 mM for 1 min at 37°C. RNAs in each quenched reaction sample were separated by PAGE (15%; 19:1 acrylamide:bis-acrylamide) in 44 mM Tris-borate, pH 8.3, 1.25 mM Na 2 EDTA, 8 M urea. The gel was exposed to a PhosphorImager screen, and the screen was scanned using Typhoon PhosphorImager software and quantified in ImageQuant. The averaged fraction of RNA at the position of the pause over time was fit to single- or double-exponential decay functions in KaleidaGraph to estimate pause efficiencies (amplitudes) and rate constants of pause escape. All pause kinetic parameters were determined from replicate (n ≥ 3) assays using error-weighted (SD) fits. For experiments comparing RNAPs, wild-type and variant RNAPs were purified side-by-side to avoid variable effects of different RNAP preparations on pausing kinetics. Rapid quench-flow measurement of nucleotide addition To measure rates of C17 and G18 addition, G16 ECs were formed essentially as described for the pause transcription assay, but with 5′-[ 32 P]RNA limiting such that RNA:tDNA:RNAP:ntDNA was 1:1.3:2:3.3. To obtain nucleotide addition rates using a quench-flow apparatus (RQF-3; KinTek Corporation, Snow Shoe, PA), 400 nM G16 ECs were injected in one sample loop and 200 µM each CTP and GTP in TB1, in the other sample loop. Reactions were performed at 37°C for the designated times and quenched with 2 M HCl, then neutralized immediately to pH 7.8 with 3 M Tris base (supplemented with 250 µg torula yeast RNA/mL). RNA products were purified by phenol:chloroform extraction followed by ethanol precipitation, and resuspended in 1X stop buffer to a constant specific activity. RNA products from all timepoints were resolved by denaturing PAGE and quantified as described for pause transcription assays. Reaction progress curves were generated for each RNA length (G16, C17 + , and G18 + ) using KaleidaGraph (Synergy Software) by calculating the fraction of total RNA for each condition as a function of time. C17 and all RNAs longer than C17 were combined to give the C17 + fraction; G18 and all RNAs longer than G18 were combined to give the G18 + fraction. The averaged fraction at each time-point was then fit to a single-exponential equation.

Kinetic modeling

To test whether the elemental pause is on online or offline state (i.e., involves a linear or branched kinetic mechanism; Figure 1—figure supplement 1F,G ) and to test whether the slow fraction of ePECs was evident at only the first or at both pause sites on the tandem pause scaffold ( Figure 1—figure supplement 2 ), we used kinetic modeling by numerical integration of pre-steady state rate equations using the program KinTek Explorer v6.1 (KinTek Corp., Snow Shoe, PA; Johnson et al., 2009 ). In both cases, to test whether the simpler mechanism was adequate to explain the data, we used replicate datasets (triplicate or greater) to generate a kinetic model for the rates of arrival at the pause site using the rate at which all RNAs before the pause site converted to RNAs at the pause site and beyond. We then held these rates constant and tested the simple kinetic models (linear, online pause ( Figure 1—figure supplement 1F,G ) or two populations of RNAP, fast and slow pausing ( Figure 1—figure supplement 2D,E ), including error for replicates to obtain the best fit and the residuals between the best fit and the observed data. We concluded that the more complex models (branched for Figure 1—figure supplement 1F,G or dynamic formation of the slow pause species for Figure 1—figure supplement 2D,E ) were favored because the residuals for the simpler mechanism exhibited large, systematic variations whereas the more complex mechanisms exhibited smaller, random variations. We did not attempt to determine which mechanism best fit the data, and limited our conclusion to rejection of the simpler mechanism. For the dataset using the template from Bochkareva et al. (2012) ( Figure 1—figure supplement 1G ), for which we had six replicates, we determined error in the fits and residuals by individually fitting each dataset and calculating the average and error for the six fits.

GreA- and GreB-stimulated cleavage assays

GreA- and GreB-stimulated cleavage and effects on pausing were assayed essentially as described earlier ( Larson et al., 2014 ). Briefly, for pause assays ( Figure 1H and Figure 1—figure supplement 3B,C ), [α- 32 P]GTP (10 µM) was maintained at constant specific activity throughout the labeling and pause assay GreA, GreB, or both (1 µM each, final) were added concurrently with the CTP and UTP (100 µM each, final). The assays were performed in triplicate and analyzed as described above. To assay the rate of cleavage product accumulation ( Figure 1I and Figure 1—figure supplement 3B,D ), accumulating small RNAs were quantified from short phosphorimager screen exposures to avoid saturating the signal.

Intrinsic cleavage assay

Intrinsic cleavage was assayed essentially as described earlier ( Mishanina et al., 2017 ). Briefly, 3′-end labeled C17 ePECs (#9563 NT DNA, #8334 T DNA, #8401 RNA; Supplementary file 1 ) were formed and immobilized on Ni 2+ -NTA beads, then washed to remove unincorporated [α- 32 P]CTP. Cleavage was initiated at 37°C by resuspending washed ePECs with Cleavage Buffer (CB; 25 mM Tris·HCl pH 9.0, 50 mM KCl, 20 mM MgCl 2 , 1 mM DTT, 5% glycerol, and 25 μg acetylated BSA/mL), and samples were collected at designated timepoints by mixing with 2X stop buffer. Cleavage products were separated by denaturing PAGE as described for transcription pause assays.

Stopped-flow fluorescence translocation assay

To measure translocation rates of the hybrid ( Figure 3 ), we used the assay developed by Belogurov and co-workers ( Malinen et al., 2012 ; Malinen et al., 2014 ). PAGE-purified template DNA and RNA were annealed in TB1 (see Supplementary file 1 ). This scaffold was incubated with RNAP for 15 min at 37°C in TB, then non-template DNA was added such that the final ratio of tDNA:RNA:RNAP:ntDNA was 1:2:3:5 (2 µM: 4 µM: 6 µM: 10 µM, respectively). This solution was diluted to 0.4 µM RNAP with TB1. ECs were then injected into one loading syringe of a stopped-flow apparatus (SF-300X; KinTek Corporation, Snow Shoe, PA) and 200 µM CTP in TB1 was loaded in the other syringe. Upon initiating rapid mixing at 37°C, 6-MI fluorescence was excited at 340 nm (2.4 nm bandwidth), and emission was monitored in real time through a 400 nm long-pass filter (Edmund Optics Inc., Barrington, NJ). The kinetics of 6-MI fluorescence unquenching was determined by fitting the average fluorescence (n ≥ 6 traces), normalized from 0 to 1, to a double exponential Equation 1 : (1) F t = A ( 1 − e − k 1 , o b s t ) + B ( 1 − e − k 2 , o b s t ) where, t = time (s); A = fast kinetic species signal amplitude; B = slow kinetic species signal amplitude; k 1,obs = observed rate of fluorescence increase for the fast kinetic species; k 2,obs = observed rate of fluorescence increase for the slow kinetic species. The fast species rate is reported as the translocation rate ( Malinen et al., 2012 ). Equilibrium fluorescence measurement of translocation To measure equilibrium translocation of downstream DNA ( Figure 4 ), G16 ECs were formed in TB1 as described for the quench-flow experiment but without Mg(OAc) 2 to stabilize ECs and with the fluorescent ntDNA as the limiting component (ntDNA:tDNA:RNA:RNAP = 1:1.5:2:3 with ntDNA at 300 nM). For equilibrium measurements of hybrid translocation ( Figure 3 ), G16 ECs were formed similarly but with the tDNA as the limiting component (see description in stopped-flow assay section). Fluorescence measurements were conducted using a PTI-spectrofluorometer (Model QM-4/2003, Photon Technology International) with 5 mM path length and 45 uL quartz cuvettes (Hellma Analytics, Müllheim, Germany). Emission spectra were obtained by exciting at 340 nm (5 nm bandwidth) and monitoring fluorescence between 360 and 500 nm (5 nm bandwidth). For each substrate addition experiment, 60 µL EC was added to the cuvette and incubated for 2 min at 37°C in the cuvette holder before performing an emission scan (average of 3 traces at 0.25 nm step size). Then, NTPs were added (concurrently with 5 mM Mg(OAc) 2 plus additional Mg(OAc) 2 equal to the NTP concentration in the assay) and the incubation was continued for 1 min (5 min for 2′dCTP) at 37°C before performing an emission scan. Between fluorescence measurements, an aliquot was removed to 2X stop buffer to subjected to denaturing PAGE to confirm nucleotide addition. For the GTP titration condition, aliquots were only removed to confirm initial G16 RNA, 3′dC addition, and failure of 20 mM GTP to incorporate following 3′dC addition. 6-MI fluorescence was quantified at 425 nm. Background fluorescence from a GTP contaminant was subtracted using signal from GTP alone in buffer. We found that the level of fluorescence from the contaminant varied significantly among vendors and GTP lot, with GTP from GE Healthcare containing the least amount. Reported increases in fluorescence are fold changes relative to the initial signal in G16 EC. Cys-pair reporter (CPR) crosslinking assays CPR crosslinking assays ( Figure 5 ) were performed as described previously ( Nayak et al., 2013 ). Nucleic acid scaffolds were prepared by annealing RNA, template DNA (T-DNA) and 15 μM non-template DNA (NT-DNA) at 10 µM, 12 µM, and 15 µM final concentrations, respectively, in reconstitution buffer (RB; 20 mM Tris-HCl pH 8, 20 mM NaCl, and 1 mM EDTA). ECs, ePECs, and his PEC were formed by incubating 1 μM RNAP and scaffold (2 μM, based on RNA) in buffer A (50 mM Tris-HCl pH 8, 20 mM NaCl, 10 mM MgCl 2 , 1 mM EDTA, and 2.5 ug acetylated bovine serum albumin/mL for 15 min at room temperature (RT). For crosslinking reactions with NTP, 3′deoxyECs formed by reaction with 3′dNTP were incubated for 15 min at RT with 0, 0.005, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5, and 10 mM GTP or ATP. Next, EC, ePEC, or his-PEC (final RNAP 0.8 uM and scaffold 1.6 uM) were incubated for 60 min with 2.5 mM CSSC and 0.05 mM DTT (E = –0.16) and stopped with 50 mM iodoacetamide. Samples were separated by native PAGE to verify reconstitution efficiency and by sodium dodecyl sulfate (SDS)-PAGE using 4–15% GE Healthcare PhastGel to quantify formation of crosslinks. Gels were stained with Coomassie Blue and imaged with a CCD camera. The fraction cross-linked was quantified with ImageJ software. Pause assays with CPR RNAPs For crosslinked CPR transcription assays, nucleic-acid scaffolds containing RNA and template DNA (1:2 ratio of RNA to DNA) were used to reconstitute ePECs or control his PECs ( Figure 6C and D ) as described in Kang et al., 2018a . The U15 ePECs containing limiting CPR RNAP (1 μM) were reconstituted on 2 µM scaffold (based on RNA) for 15 min at 37°C in Elongation Buffer (EB; 25 mM HEPES-KOH, pH 8.0, 130 mM KCl, 5 mM MgCl 2 , 1 mM DTT, 0.15 mM EDTA, 5% glycerol, and 25 μg acetylated bovine serum albumin/mL), followed by addition of 6 μM non-template DNA and further incubation for 10 min at 37°C to complete assembly of the transcription complexes. Wild-type RNAP was tested as a control side-by-side with CPR RNAPs. Crosslinking of 1 μM ePECs was performed in the presence of 1 mM cystamine as the oxidant and 0.8 mM DTT, for 15 min at 37°C. An aliquot of the crosslinking reaction was quenched with 15 mM iodoacetamide (final concentration) and analyzed by non-reducing SDS-PAGE to confirm formation of the crosslink. The crosslinked U15 ePECs were diluted to 0.2 μM with EB (without DTT, for crosslinked samples) and incubated with heparin (0.1 mg/mL final) for 3 min at 37°C. The U15 ePECs were then radiolabeled by extension with 20 μM [α- 32 P]GTP for 1 min at 37°C, to poise the complexes one nucleotide before the pause sequence. The resulting G16 ePECs were further diluted to 0.1 μM (based on RNAP) and assayed at 37°C for pause-escape kinetics at 10 µM GTP by addition of CTP in EB to 100 µM (without DTT, for crosslinked samples). Reaction samples were removed at various time points and quenched with an equal volume of 2X stop buffer. All active ePECs were chased out of the pause with 500 μM GTP and CTP, each, for 5 min at 37°C. RNAs in each quenched reaction sample were separated on a 15% urea-PAGE gel. Gels were visualized and quantified as described for in vitro transcription assays. Cys Triplet Reporter assays For Cys triplet reporter (CTR) cross-linking assays ( Figure 6B,E and F ), ECs and PECs were assembled on purified DNA and RNA scaffolds specified in the figure legend and as described previously ( Kang et al., 2018a ). Briefly, 10 μM RNA, 12 μM template DNA, and 15 μM non-template DNA ( Supplementary file 1 ) were annealed in RB. To assemble complexes, scaffold (2 μM) was mixed with limiting CTR RNAP (1 μM; CTR RNAP: β′1045iC 258iC, β843C) in 50 mM Tris-HCl, pH 7.9, 20 mM NaCl, 10 mM MgCl 2 , 0.1 mM EDTA, 5% glycerol, and 2.5 μg of acetylated bovine serum albumin/mL, and added to mixtures of cystamine and DTT to generate redox potentials that ranged from −0.314 to −0.424. Complexes were incubated for 60 min at room temperature and then were quenched with the addition of iodoacetamide to 15 mM. The formation of cysteine-pair cross-links was then evaluated by non-reducing SDS-PAGE (4%–15% gradient Phastgel; GE Healthcare) as described previously ( Nayak et al., 2013 ). Gels were stained with Coomassie Blue and imaged with a CCD camera. The fraction cross-linked was quantified with ImageJ software. The experimental error was determined as the standard deviation of measurements from three or more independent replicates.

Reagents and materials

Plasmids and oligonucleotides are listed in Supplementary file 1 . RNA and DNA oligonucleotides were obtained from Integrated DNA Technologies (IDT; Coralville, IA) and purified by denaturing polyacrylamide gel electrophoresis (PAGE) before use. GreA, GreB, and RNAPs were purified as described previously ( Larson et al., 2014 ; Windgassen et al., 2014 ). Briefly, His-tagged RNAPs were overexpressed in E. coli BL21 λDE3 and cells were lysed by sonication. RNAPs were enriched by PEI and ammonium sulfate precipitation, then purified by sequential nickel (5 mL HisTrap) and heparin (5 mL HiTrap) column chromatography, dialyzed into storage buffer (20 mM Tris-Cl, pH 8, 250 mM NaCl, 20 μM ZnCl 2 , 1 mM MgCl 2 , 0.1 mM EDTA, 1 mM DTT, and 25% glycerol), and stored in small aliquots at –80° C.

Additional files 10.7554/eLife.40981.022 Supplementary file 1. Oligonucleotides and plasmids used in this study. 10.7554/eLife.40981.023 Transparent reporting form Data availability All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 2 and 7.