Chemistry

Single-molecule characterization of extrinsic transcription termination by Sen1 helicase

Plasmids and sequences

A 2-kb DNA fragment was PCR amplified from the Thermus aquaticus RPOC gene, with primers RPOC_F and RPOC_R (see primer checklist in Supplementary Desk) containing, respectively, an XbaI and an SbfI web site individually. This fragment accommodates a singular, centrally situated, and distinctive KpnI web site for insertion, in later cloning steps, of particular transcription models. This 2-kb fragment was subcloned by the XbaI and SbfI websites into the pUC18 polylinker to construct the spine plasmid. The distinctive EcoRI of pUC18 was destroyed by first digesting with EcoRI, then blunting the overhang with the Fast Blunting Package (NEB), and at last ligating the blunt ends along with the Fast Ligation Package (NEB) previous to cloning.

We designed and obtained by way of gene synthesis (Eurofins Genomics) the DNA sequence Pol2-144-444-his (see Supplementary Notes) and which is flanked by KpnI websites (pink). This sequence presents a area delimited by distinctive HindIII (blue) and SpeI (inexperienced) websites and into which one can insert the completely mispaired bubble used as a scaffold for Pol II initiation. This modular area is flanked on both facet by the his transcription termination sequence of E. coli (underlined). The sequences to be transcribed and that are contained between the HindIII and his terminator on one facet, and the SpeI and his terminator on the opposite facet, are from the E. coli lacZ gene and are 144 and 444 bp lengthy, respectively. The Pol2-144-444-his fragment was cloned into the spine plasmid by way of its KpnI websites. The ensuing DNA plasmid, named pUC18 Pol2-144-444-his, was purified from an in a single day tradition of E. coli utilizing the Nucleobond Xtra Midi Plus equipment for alkaline lysis and ion trade (Macherey-Nagel).

We designed and obtained by way of gene synthesis (Eurofins Genomics) the DNA sequence Pol2-444-444-his (see Supplementary notes). This sequence presents a area delimited by distinctive HindIII (blue) and SpeI (inexperienced) websites, and into which one can insert the completely mispaired bubble used as a scaffold for Pol2 initiation. It differs from the Pol2-144-444-his sequence described above in that each sequences to be transcribed and that are contained between the HindIII web site and his terminator on one facet, and the SpeI web site and his terminator on the opposite facet, are 444 bp lengthy. Distinctive AvrII (daring, mild inexperienced) and MluI (daring, purple) restriction web site are additionally current on this sequence for added modularity. This fragment was cloned into the spine plasmid by way of its KpnI websites. The ensuing DNA plasmid, named pUC18 Pol2-444-444-his, was purified from an in a single day tradition of E. coli utilizing the Nucleobond Xtra Midi Plus equipment for alkaline lysis and ion trade (Macherey-Nagel).

We constructed the DNA sequence Pol2-806-806-his by way of the next process. We first PCR amplified two 362 bp fragments from Pol2-444-444-his plasmid utilizing the primers 444_LF and 444_LR (containing, respectively, distinctive AvrII and SpeI websites proven underlined) and 444_RF and 444_RR (containing, respectively, distinctive AscI and MluI websites proven underlined).

The primary PCR fragment was digested with AvrII and SpeI and cloned into the pUC18 Pol2-444-444-his plasmid by way of by its AvrII web site. The specified orientation of the 362 bp fragment was chosen by sequencing and maintained the AvrII web site on the 5′ finish of the sequence. Then, the second PCR fragment was digested with AscI and MluI and cloned into the pUC18 Pol2-444-444-his plasmid by its MluI web site. The specified orientation of the 362 bp fragment was chosen by sequencing and maintained the MluI web site on the three′ finish of the sequence. The ensuing plasmid, named pUC18 Pol2-806-806-his, was expressed and purified as above.

The Pol2-G-less-cassette sequence specified beneath was designed and obtained by gene synthesis (see Supplementary notes). It accommodates two KpnI websites (pink) and a area flanked by HindIII (blue) and EcoRI (yellow) websites. The sequence additionally accommodates distinctive SpeI (inexperienced) and MluI (daring, purple) websites. Downstream of the EcoRI web site is a G-less sequence. This sequence was cloned into the spine plasmid by its KpnI websites. The ensuing plasmid, named pUC18 Pol2-G-less cassette, was expressed and purified as above.

We designed and obtained by way of gene synthesis (Eurofins Genomics) the DNA sequence T5N25-178his (see Supplementary notes). This sequence presents a T5N25 promoter sequence (blue) adopted by 178 bp transcription unit and a his terminator. This fragment was cloned into the spine plasmid by way of its KpnI websites. The ensuing DNA plasmid, named pUC18 T5N25-178his, was purified from an in a single day tradition of E. coli utilizing the Nucleobond Xtra Midi Plus equipment for alkaline lysis and ion trade (Macherey-Nagel).

DNA constructs for tethered-DNA supercoiling assays

We ready three constructs containing a scaffold bubble flanked by a his terminator on either side. The Pol2-144-444-his assemble sustains bidirectional transcription from the bubble; in a single path an E. coli his terminator lies 144 bp away; within the different path a his terminator lies 444 bp away. The Pol2-444-444-his assemble differs in that the his terminators are every situated 444 bp from the bubble, whereas for the Pol2-806-806-his assemble the his terminators are every situated 806 bp from the bubble.

First, plasmids pUC18 Pol2-144-444-his, pUC18 Pol2-444-444-his, and pUC18 Pol2-806-806-his had been digested three h with HindIII and SpeI restriction enzymes (New England Biolabs). The ~20 bp fragment contained between the HindIII and SpeI websites was eliminated by agarose gel electrophoresis and the longer (~5 kb) fragment extracted from the gel (Macherey-Nagel PCR and Gel Extraction Package).

5′ phosphorylated oligonucleotides non-tem1 and tem1 (Eurofins Genomics) had been annealed to type a scaffold dsDNA oligo by combining to 50 μM every in 1x PBS, heating to 95 oC for two min, after which cooling to room temperature over a 2 h interval. The areas underlined within the oligos type an unpaired area after annealing the scaffold, and the ends of the annealed scaffold oligo are suitable with ligation into HindIII and SpeI overhangs.

The Pol2-144-444-his, Pol2-444-444-his, and Pol2-806-806-his constructs for tethered-DNA supercoiling assays had been ready by in a single day ligation at room temperature of the scaffold oligo into the pre-digested and purified pUC18 Pol2-144-444-his, Pol2-444-444-his, or Pol2-806-806-his plasmids. The ligation product was then purified with a NucleoSpin Gel and PCR Clear-Up equipment (Macherey-Nagel) after which digested with XbaI, SbfI, and AseI (New England Biolabs). The specified DNA fragment (2.7 kb for the Pol2-144-444-his assemble, three kb for the Pol2-444-444-his assemble, and three.four kb for Pol2-806-806-his assemble) was remoted by gel purification and extraction. The DNA molecules had been then ligated to 1 kb DNA fragments modified with a number of biotin teams by the XbaI web site and to 1 kb DNA fragments modified with a number of digoxigenin teams by the SbfI web site. The modified DNA fragments had been synthesized by way of PCR amplification within the presence of dUTP-biotin and dUTP-digoxigenin, respectively (Strick 2005 nmeths).

The T5N25-178his assemble was ready by digesting the T5N25-178his plasmid with XbaI, SbfI, and AseI (New England Biolabs) and isolating by way of gel purification and extraction. The produced 2.2 kb DNA had been then ligated to 1 kb DNA fragments modified with a number of biotin teams by the XbaI web site and to 1 kb DNA fragments modified with a number of digoxigenin teams by the SbfI web site.

Stalling constructs for tethered-DNA supercoiling assays

Two constructs containing a scaffold bubble and a G-less transcription cassette had been ready. The Pol2-G-less-137 assemble sustains unidirectional transcription from the scaffold bubble and stalls Pol2 137 bp from the bubble. The Pol2-G-less-19 assemble sustains unidirectional transcription from the scaffold bubble and stalls Pol2 19 bp from the bubble.

First, plasmid pUC18 Pol2-G-less cassette was digested three h with HindIII and EcoRI restriction enzymes (New England Biolabs). The ~20 bp fragment contained between the HindIII and EcoRI websites was eliminated by agarose gel electrophoresis and the longer (~5 kb) fragment extracted from the gel (Macherey-Nagel PCR and Gel Extraction Package).

5′ phosphorylated oligonucleotides non-tem1 and tem2 (Eurofins Genomics) had been annealed to type a second scaffold dsDNA oligo as above. The areas underlined within the oligos type an unpaired area after annealing the scaffold, and the ends of the annealed scaffold oligo are suitable with ligation into HindIII and EcoRI overhangs.

The 2 stalling constructs (Pol2-G-less-137 and Pol2-G-less-19) for tethered-DNA supercoiling assays had been ready similarly. For the Pol2-G-less-137 assemble, the second scaffold oligo (non-tem1 and tem2) was ligated into the pre-digested Pol2-G-less-cassette plasmid ready as above. To assemble the Pol2-G-less-19 assemble, the primary scaffold oligo (non-tem1 and tem1) was ligated into Pol2-G-less-cassette plasmid pre-digested as above however with the HindIII and SpeI restriction enzymes. Remaining steps of the meeting process had been carried out as described above.

Stalling constructs for tethered-Pol II translocation assays

The Pol2-G-less-137-T stalling assemble for tethered-Pol II translocation assays was ready following the identical procedures as Pol2-G-less-137 assemble preparation, as much as and together with in a single day ligation of the scaffold bubble into the plasmid and purification of the ligation response from enzymes utilizing a Macherey-Nagel PCR and Gel Extraction Package. The purified ligation product was then digested with XbaI and NcoI (the NcoI web site is situated between the XbaI web site and the inserted bubble scaffold) and the goal DNA fragments (~four.6 kb) was remoted by gel purification. The goal DNA molecules had been then ligated to 1 kb DNA fragments modified by a number of digoxigenins by way of the XbaI web site and within the presence of NcoI-restriction enzyme. When the ensuing DNA is tethered to an antidigoxigenin-treated glass floor by way of the digoxigenin teams, transcription from the scaffold directs Pol II in direction of the glass floor.

2-mer RNA (5′-GpA) was bought from TriLink Bio Applied sciences and 9-mer RNA (5′-ACACGGCGA) was from Dharmacon/GE Healthcare.

Surfaces used for single-molecule experiments had been ready and derivatized with anti-digoxigenin28.

Tethered-DNA supercoiling assays

DNA molecules had been first hooked up to 1-μm-diameter streptavidin-coated superparamagnetic beads (Dynabeads MyOne Streptavidin C1; Life Applied sciences) after which to a glass floor functionalized with anti-digoxigenin. The glass floor was positioned atop a home made magnetic lure which screens and analyzes the place of the tethered superparamagnetic bead with the PicoJai software program bundle (PicoTwist SARL). Knowledge had been collected at video charge (31.0802 Hz) and filtered at zero.5 Hz. The usual deviation for bead fluctuations at 31 Hz was s = 20 nm and the bead cutoff frequency was four Hz. Knowledge had been processed utilizing customized routines within the Xvin software program subsuite of PicoJai. Experiments had been carried out in normal transcription buffer containing 20 mM Okay-HEPES pH 7.5, 150 mM Okay-Glut, eight mM Mg(Ac)2, zero.5 mg/ml BSA, zero.1% w/v Tween 20, 2 mM DTT, and 10 μM ZnCl2 at 28 °C. DNA molecules had been prolonged and torsionally constrained (F = zero.three pN, the place 1 pN = 10−12 N; superhelical density = −zero.027 or −7 turns for unfavorable supercoiling; zero.zero16 or +four turns for constructive supercoiling).

Steady-tracking and pulse-chase methodologies had been used for tethered-DNA supercoiling assays, displaying equivalent outcomes when it comes to quantitative evaluation and thus merely representing totally different ranges of optimization of those measurements8,15.

Steady monitoring methodology was used to check Pol II transcription initiating from the bubble, by which Pol2-144-444-his assemble was used for testing Pol II initiates and transcribes in both path from the bubble by addition of 1 nM Pol II, 1 μM 2-mer RNA, 1 mM NTPs containing 1 mM every of ATP, UTP, GTP, and CTP, and 1 nM TFIIS. Pol2-144-444-his assemble, Pol2-444-444-his assemble and Pol2-806-806-his assemble had been used for testing transcription size results on Pol II transcription, by which 25 nM TFIIS was used. NTPs concentrations (zero.1, zero.2, and 1 mM) had been titrated through the use of Pol2-144-444-his assemble. For testing TFIIS actions, the Pol2-444-444-his assemble was used with zero or 25 nM TFIIS. For Sen1 termination experiments, Pol2-G-less-137 stalling assemble, 1 nM Pol II, 1 μM 2-mer RNA, 2 mM ATP, and 1 mM every of UTP and CTP, 1 nM TFIIS, and varied Sen1 HD concentrations (10, 20, 40, and 100 pM) had been used.

The heartbeat-chase methodology has been described beforehand17. Single-round ‘pulse-chase’ assays are optimum for measuring the interactions of associate proteins with a single RNA polymerase stalled on DNA, with out interference or added measurement noise generated by free RNA polymerase in resolution. To stall Pol2 on nanomanipulated, supercoiled DNA, we first added 1 nM Pol II, 1 μM 2-mer RNA, 1 mM AUC, and 1 nM TFIIS and incubated for 2000 s. Longer incubation time was used to stall a number of Pol II molecules on the identical DNA assemble. We then washed away free elements with transcription buffer, after which added 500 pM Sen1 FL or 40 pM Sen1 HD, together with 1 mM ATP, to measure the displacement of stalled Pol2. For measurements assaying the function of ATP binding and hydrolysis in Sen1 exercise, 40 pM Sen1 HD was added to stalled Pol2 both within the absence of ATP or within the presence of 1 mM ATPγS. Constructive controls of unfavorable controls had been carried out on the identical molecules by ultimate addition of 40 pM Sen1 HD and 1 mM ATP.

For the RNaseA assays, we assembled reactions on the Pol2-G-less-cassette assemble by mixing 1 nM Pol II + AUC (1 mM every), 40 pM Sen1 HD + ATP (1 mM), and RNase A (zero, 50, or 100 μg/ml, Thermo Fischer).

Tethered-Pol II translocation assays

Pol II was stalled on the Pol2-G-less-137-T assemble at +137 (the primary hybridized base pair downstream of the bubble was counted as +1) by incubating eight pM DNA with 1 nM biotin-labeled Pol II, 1 μM 2-mer RNA, 1 mM AUC containing 1 mM every of ATP, UTP, and CTP, and 1 nM TFIIS at 28 °C for 30 min; adopted by mixing with streptavidin-coated beads and deposition onto the glass floor. After washing away free elements, a 1 pN drive was utilized to softly lengthen the DNA molecules. Pol2 transcription was restarted by including the 4 nucleotides (1 mM every) alone or with 1 nM TFIIS. For Sen1 termination experiments 500 pM Sen1 HD or Sen1 FL had been added together with 1 mM ATP.

Time-traces had been analyzed utilizing the PicoJai software program suite (PicoTwist SARL) and transcription pulses had been manually assigned and analyzed for period and amplitude.

Bulk assays

DNA oligos (non-tem3 and tem3, Eurofins Genomics, FPLC purified, see Supplementary Desk) had been blended in equimolar quantities (10 μM every) in 10 mM Tris pH 7.5, 50 mM NaCl buffer, heated to 95 °C for two min and cooled slowly to room temperature to type the transcription template containing a everlasting unpaired area (or bubble, highlighted in yellow). A ultimate focus of zero.1 mg/ml BSA was added after annealing the duplex.

Remaining concentrations of 5 nM annealed duplex, 50 nM Pol2, 10 μM UTP, zero.33 μM α-radiolabeled UTP, 1 mM ATP + CTP or 1 mM ATP + GTP + CTP, and 1 μM GpA had been incubated in transcription buffer (20 mM Tris–HCl pH 7.5, 100 mM NaCl, eight mM MgCl2, 10 μM ZnCl2, 10% (v/v) glycerol, 2 mM DTT) at 28 °C for 20 or 60 min for Pol2 transcription. After the response, the radiolabeled transcript was migrated on denaturing polyacrylamide sequencing gels.

Protein purification

S. cerevisiae RNA Polymerase II (Pol II) was purified from a pressure that expresses a His6-tagged model of Rpb3p primarily as beforehand described6. Briefly, the cell pellet was resuspended in lysis buffer (20 mM Tris–HCl pH eight, 150 mM KCl, 10% (v/v) glycerol, 10 mM ZnCl2, 10 mM DTT) and lysed utilizing a Carver press. After clarification, the protein extract was precipitated with 40% ammonium sulfate, and subjected to Ni-affinity chromatography (Ni-NTA, Qiagen) after which anion trade chromatography (Mono-Q 5/50 GL, GE Healthcare). The fractions of curiosity had been dialyzed towards Pol2 storage buffer (10 mM HEPES pH 7.9, 40 mM (NH4)2SO4, 10 mM ZnCl2, 10% (v/v) glycerol, 10 mM DTT) and saved at −80 °C.

Biotinylated RNA Pol II was ready by incubating 50 mg of Pol II bearing the AviTag and 10 µg of BirA biotin ligase protein in 200 µl response buffer (10 mM HEPES pH 7.9, 40 mM (NH4)2 SO4, 5 µM ZnCl2 2.5 mM DTT, 5% (v/v) glycerol, 10 mM MgAc-ATP, and zero.1 mM biotin) for five h at four °C, adopted by dialysis towards Pol2 storage buffer and saved at −80 °C.

E. coli RNA polymerase (ecRNAP) was purified as beforehand described16. Briefly, the cell pellet was resuspended in lysis buffer (20 mM Tris–HCl, pH eight.zero, 500 mM NaCl, 5% glycerol) and lysed utilizing Emulsiflex C5, Avestin. After clarification, the protein was loaded onto 10 ml of nickel-chelated metal-affinity resin (HiTrap Chelating, GE Healthcare) after which subjected to 10 ml of heparin resin (HiTrap Heparin, GE Healthcare). The fractions containing core RNAP had been pooled and half the pooled quantity was saturated with recombinant σ70 (ready as in ref. 29) and dialyzed in a single day into dialysis buffer (20 mM Tris–HCl, pH eight.zero, 200 mM NaCl, zero.1 mM EDTA, 1 mM DTT, and 50% glycerol) earlier than flash freezing and storage at −80 °C (to make holoenzyme ecRNAP). The opposite half-volume was not saturated with σ70 (i.e. core ecRNAP) however immediately aliquoted, flash frozen, and saved at −80 °C.

Sen1 FL was purified from yeast pressure DLY1774 (derived from W303) as beforehand described5, which overexpresses N-terminal TAP-tagged Sen1 FL from the GAL1 promoter. Cell pellet from four l of YPA tradition containing 2 g/l of galactose at OD600 ≈ 2 was resuspended in AGK buffer (10 mM HEPES pH 7.9, 1.5 mM MgCl2, 200 mM KCl, 10% (v/v) glycerol, zero.5 mM DTT) containing protease inhibitors (2 mM AEBSF, 2 mM benzamidine, and EDTA-free Protean from Roche) and lysed utilizing a Retsch MM301 Ball Mill. The suspension was clarified by centrifugation (30 min at 30,000×g at four °C) and handled with RNaseA + T1 (10 mg/ml) for 20 min at 25 °C earlier than loading onto IgG-sepharose beads (GE Healthcare) pre-equilibrated with IPP150 buffer (10 mM Tris–HCl pH 7.5, 150 mM NaCl, zero.1% NP40, 5% (v/v) glycerol). Beads had been profusely washed with IPP150 after which with IPP500 (as IPP150 however containing 500 mM NaCl) after which with TEV cleavage buffer (10 mM Tris–HCl pH 7.5, 150 mM NaCl, zero.1% NP40, zero.5 mM EDTA, 5% (v/v) glycerol, 1 mM DTT) earlier than in a single day incubation with TEV protease at four °C. Protein launched from the beads was subjected to additional purification by calmodulin-affinity chromatography after which dialyzed towards storage buffer (10 mM Tris–HCl pH 7.5, 150 mM NaCl, 50% (v/v) glycerol, 1 mM DTT) and saved at −80 °C.

His8-CPD-tagged Sen1 HD (1095–1904) was produced as before6. Briefly, Sen1 HD with a cleavable C-terminal His-tag coupled to Vibriocholerae MARTX toxin cysteine protease area was purified from Escherichia coli BL21 (DE3) STAR pRARE (Stratagene) cells grown in TB medium. Cells had been lysed in buffer containing 20 mM sodium phosphate pH eight.zero, 500 mM NaCl, 2 mM MgCl2, 30 mM imidazole, 10% (v/v) glycerol, 1 mM β-mercaptoethanol, benzonase, and protease inhibitors, and sure to a Ni2+-affinity chromatography column (HisTrap FF, GE Healthcare) adopted by on-column tag cleavage utilizing 3C protease30. The samples had been utilized to a HiTrap Heparin HP column (GE Healthcare) equilibrated in buffer A (20 mM Tris–HCl pH 7.5, 200 mM NaCl, 2 mM MgCl2, 1 mM DTT) and eluted by growing a gradient to 1 M NaCl. Dimension-exclusion chromatography was carried out utilizing a Superdex HiLoad 200 column (GE Healthcare) equilibrated in GF buffer (20 mM HEPES pH 7.5, 300 mM NaCl, 2 mM MgCl2, and 1 mM DTT, 50% (v/v) glycerol) and the purified proteins had been saved at −80 °C.

TFIIS was purified as described24 by way of nickel-affinity chromatography. Peak fractions had been pooled and diluted five-fold with Mono-S buffer A (50 mM HEPES pH 7.5, zero.01 mM ZnCl2, 1 mM DTT, and 10% (v/v) glycerol) and loaded onto a Mono-S anion trade column (GE Healthcare) equilibrated in Mono-S buffer A. TFIIS was eluted from the Mono-S column by growing a gradient to 1 M NaCl. The second peak fraction was collected and concentrated to ~1 ml (VivaSpin, three kDa MWCO, GE Healthcare) after which gel-filtrated (Superdex HiLoad 200 16/60, GE Healthcare) in GF buffer (25 mM HEPES pH 7.5, 250 mM NaCl, zero.01 mM ZnCl2, 10 mM DTT, and 10% (v/v) glycerol) earlier than aliquoting and shock-freezing in liquid nitrogen. Single-use aliquots had been saved at −80 °C.

Hexahistidine-tagged BirA was purified from three l of IPTG-induced BL21 (DE3) tradition utilizing Ni-affinity chromatography (His-Lure, GE Healthcare). Briefly, cell pellet was resuspended in lysis buffer (25 mM Tris–HCl pH eight, 250 mM NaCl, 5% (v/v) glycerol) supplemented with EDTA-free Full Protease Inhibitor (Roche) and lysed utilizing an Avestin C5. After clarification, supernatant was loaded onto 10 ml of nickel-bound metal-chelating resin (HiTrap Chelating column, GE Healthcare). Contaminants had been eliminated by washing resin with 50 mM imidazole, and BirA was eluted by growing an imidazole gradient to 1 M. Protein-containing fractions had been pooled and imidazole was eliminated by buffer trade (G25 Desalting Column, GE Healthcare) towards buffer containing 20 mM Tris–HCl pH eight, 200 mM NaCl, and 5% (v/v/) glycerol. Protein was aliquoted, flash frozen, and saved at −80 °C.


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