Ensemble studies of transcription in vitro have provided a wealth of knowledge on transcript elongation by eukaryotic RNAP II and its modulation by elongation factors.1-4 However, the mecha-nisms by which the DNA template, RNA transcript and protein factors alter the active-site configuration of RNAP II to affect its elongation properties remain under investigation. At every nucleotide addition step the active center of RNAP II must move between pre- and post-translocated states,5-8 a process that could be regulated by the nucleic acid sequences surrounding the site of nucleotide addition, by protein factors, or by both. A population of RNAP II molecules, even if started simultaneously at the same nucleotide position, rapidly loose synchrony due to the stochas-tic distribution of elongation rates, possible heterogeneity among subsets of RNAP II molecules and variable pause efficiencies at some template positions. Further, the rapid rate of translocation between pre- and post-translocated states is difficult to resolve by ensemble measurements.
Single-molecule optical-trapping methods have yielded sig-nificant insights in the mechanism of elongation by bacterial RNAP.9-18 These techniques provide superior temporal and spa-tial resolution, as well as the ability to apply assisting or hinder-ing forces that can reveal the contributions of translocation to
Single-molecule studies of RNA polymerase II (RNAP II) require high yields of transcription elongation complexes (TECs) with long DNA tethers upstream and downstream of the TEC. Here we report on a robust system to reconstitute both yeast and mammalian RNAP II with an efficiency of ~80% into TECs that elongate with an efficiency of ~90%, followed by rapid, high-efficiency tripartite ligation of long DNA fragments upstream and downstream of the reconstituted TECs. Single mammalian and yeast TECs reconstituted with this method have been successfully used in an optical-trapping transcription assay capable of applying forces that either assist or hinder transcript elongation.
Efficient reconstitution of transcription elongation complexes for single-molecule studies
of eukaryotic RNA polymerase IIMurali Palangat,1,2,* Matthew H. Larson,3,† Xiaopeng Hu,4,‡ Averell Gnatt,4 Steven M. Block5 and Robert Landick6
1Laboratory of Receptor Biology and Gene Expression; NCI; Bethesda, MD USA; 2Department of Biochemistry; University of Wisconsin; Madison, WI USA; 3Biophysics Program; Stanford University; Stanford, CA USA; 4Department of Pharmacology and Experimental Therapeutics; University of Maryland School of Medicine; Baltimore, MD USA;
5Departments of Applied Physics and Biology; Stanford University; Stanford, CA USA; 6Departments of Biochemistry and Bacteriology; University of Wisconsin; Madison, WI
†Current Affiliation: Department of Cellular and Molecular Pharmacology; University of California; San Francisco, CA; ‡Current Affiliation: School of Pharmaceutical Science; Sun Yat-sen University; Guangzhou, China
Keywords: RNA polymerase II, reconstitution, transcription elongation complexes, single molecule transcription, optical trapping
Abbreviations: RNAP II, RNA polymerase II; TEC, transcription elongation complexes; CT RNAP II, calf thymus RNA polymerase II
RNAP elongation properties at different template positions and in the presence of various protein regulators. To apply such approaches to study the mechanistic details of transcript elon-gation by eukaryotic RNAP II, a similar robust single-molecule assay system capable of studying elongation-competent RNAP II molecules from both yeast and mammalian cells is needed.
Use of single-molecule methods to study yeast RNA poly-merase19,20 was recently demonstrated. Bustamante and cowork-ers19-21 used TECs assembled on oligonucleotide scaffolds,22,23 enriched for an active fraction by a restriction endonuclease digestion, and studied elongation by individual molecules of yeast RNAP II. By separately ligating either a downstream or upstream DNA tether to the TEC, this assay allowed for the application of hindering19 or assisting loads20 on elongating yeast RNAP II molecules using an optical trap. However, low recon-stitution efficiencies of only ~10% were achieved in these stud-ies,21 significantly reducing the number of elongation-competent TECs available for characterization. This system is further lim-ited by the inability of many of the reconstituted TECs to resume elongation when supplied with NTPs.21 A reconstitution system that not only supports efficient assembly of a large fraction of elongation competent mammalian TECs, but also allows study of RNAP molecules from different organisms10,13,15 would greatly simplify single-molecule methodologies. We set out to develop a
downstream DNA allows for application of force that hinders elongation and favors backtracking (Fig. 1B). The upstream or downstream DNA can be tethered using a 5'-digoxigenin label on the DNA and anti-digoxigenin antibody coated polystyrene bead. To exert force along the RNA, the emerging transcript can be tethered via a digoxigenin-labeled DNA handle with a single stranded overhang complemen-tary to the first 25 bases of the emerging transcript (Fig. 1C). Forces in the RNA-pulling assay have the same directional sign as loads applied in the assisting-load DNA-pulling assay, leading to suppression of RNAP backtracking.10
Mammalian RNAP II can be effi-ciently reconstituted in vitro to form elongation competent TECs. We set out to develop a template system to study tran-script elongation by single molecules of CT RNAP II using an optical-trapping assay. These templates needed to be sev-eral kilobase-pairs long to allow sufficient separation of the polystyrene beads when held in the dual-beam optical trap. Such long templates are also necessary to allow adequate time for data acquisition during transcript elongation by single molecules of RNAP. We created such a template by initially reconstituting CT RNAP II on a
nucleic acid scaffold, followed by tripartite ligation of 2.7 kb and 4.5 kb DNA fragments to the upstream and downstream ends of the scaffold, respectively. In the single-molecule assay, we wished to transcribe DNA that would normally be transcribed by human or other mammalian RNA polymerases in vivo. Therefore, we chose a 4.5 kb fragment from the 5'-end of human RPB1 gene for the downstream DNA fragment.
The limited step reconstitution assay24 used previously to assemble transcriptionally active bacterial TECs was employed here to reconstitute CT RNAP II. Briefly, short template strand DNA and RNA were annealed to form a nucleic acid scaffold, which was then incubated with purified CT RNAP II to form a protein-nucleic acid binary complex (Fig. 2A). The fully com-plementary non-template strand was then added to the binary complex to form a TEC. The ability of the reconstituted TEC to elongate the RNA by 1 nt in the presence of α-32P-ATP to make A15 RNA (Fig. 2B) was used as a measure of reconstitution effi-ciency. Quantitative analysis of the accumulation of A15 RNA indicated a reconstitution efficiency of ~90%. The presence of CT RNAP II in excess of pre-formed scaffold ensured that all of the scaffolds were fully utilized. For all further experiments, the labeled A15 TECs were incubated with ATP and GTP to make the halted A20 complex. Upon addition of ATP, CTP and GTP (1 mM each), ~90% of the A20 TECs elongated efficiently and synthesized the 72mer run-off RNA from the scaffold (Fig. 2C).
system that would support robust reconstitution of mammalian RNAP II (Calf Thymus RNAP; referred to as CT RNAP II here-after) that is known to engage promoter DNA poorly and requires multiple protein regulators to initiate transcription accurately in vitro.1,2 Such a system that meets the stringent requirements of mammalian RNAP II transcription could be easily adapted for structure-function studies of protein-nucleic acid and protein-protein interactions in TECs using mutant yeast or mammalian RNAP II that cannot initiate at promoters. Here we report, for the first time, the development of a robust and efficient reconsti-tution-ligation system to assemble elongation competent mam-malian TECs to study single molecules of mammalian RNAP II using an optical-trapping assay.
Results
Template features for the single-molecule RNAP II transcrip-tion assay. In our optical-trapping assay, two distinct load con-figurations are possible based on the point of attachment of beads to the DNA template (upstream or downstream), the RNA tran-script, or RNAP II (via an anti-RPB1 antibody, 8WG16, that binds to the C-terminal domain of the RPB1 subunit of RNAP II) (Fig. 1). Attaching beads to the upstream DNA of a TEC allows application of force that assists the normal elongation motion of RNAP II (Fig. 1A), whereas attaching a bead to the
Figure 1. Template features and experimental geometry for the single-molecule RNAP II optical-trapping assay. Different templates and experimental geometries were used to measure elonga-tion under conditions of (A) assisting force along the DNA, (B) hindering force along the DNA or (C) in principle, assisting force along the RNA (not used in this study).
transcript was coded by the ligated downstream DNA fragment after the incorporation of GTP at position 76 (Fig. 4A and indi-cated as Halt #2, G76). Thus, in the presence of ATP, CTP and GTP, the elongating A20 TEC should halt at position G76 for lack of UTP if the downstream DNA was ligated to the scaffold, or would run-off at G72 if the downstream DNA was not ligated to the scaffold. A20 TECs elongated to G72 when there was no downstream DNA or to G76 in the presence of ligated downstream DNA (Fig. 4B and compare lanes 2 and 3). When UTP was added, TECs halted at G76 elongated further (Fig. 4B and lane 4). Importantly, almost all of the TECs halted at G76 elongated further (compare lanes 3 and 4). Formation of a halted G76 TEC and its elongation in the presence of UTP were dependent on the ligation of downstream DNA to the scaffold (Fig. S2). Quantitative analysis of lanes 3 and 4 (Fig. 4B) indicate that ~80% of the TECs elongate efficiently from position A20 (Fig. 4C). Only a small fraction ~8% of the TECs terminate at G72 indicating that these templates lacked the ligated downstream DNA and ~90% of the complexes that reached position G76 elongated efficiently into longer products
We included 100 mM ammonium ions in our elon-gation assays as it has been shown to enhance the rate of elongation by RNAP II in vitro.25,26 Thus, the system we established reconstituted CT RNAP II in vitro robustly and efficiently to yield highly active, elongation-competent TECs.
Electrophoretic mobility shift on gradient aga-rose gels to monitor ligation of DNA tethers. To study transcript elongation over multiple rounds of nucleotide addition steps, we created long tem-plates by ligating a 4.5 kb DNA fragment to the downstream end of the reconstituted TECs and a 2.7 kb DNA fragment to the upstream end. The DNA oligonucleotides used to reconstitute TECs were designed such that, upon annealing, the tem-plate and non-template DNA oligonucleotides created unique Sty I and Dra III restriction endo-nuclease sites at the upstream or downstream end of the duplex, respectively. The upstream and down-stream DNA tethers were cleaved with Sty I and Dra III, and then ligated to corresponding ends of the pre-assembled scaffold in the TEC. The sequence of the DNA scaffold was also designed as a U-less cassette. As such, the ligated downstream DNA codes for the first UMP base incorporated into the RNA transcript. We first established conditions for ligation of the upstream and downstream DNA to the scaffold without assembly of TECs (Fig. S1). Ligation of the DNA handles to the labeled A20 TECs was detected by electrophoretic mobility shift assay on a 1% and 4% step gradient agarose gel (Fig. 3A and B). The mobility of the labeled A20 TEC was significantly shifted based upon liga-tion of upstream (Fig. 3B and lane 3), or down-stream DNA (lane 5) with retardation dependent on the size of the fragment being ligated (compare lanes 3 and 5). Ligation of both the upstream and downstream fragments resulted in further retardation of the complex (lanes 7–9). The presence of labeled ATP from the transcription reac-tion resulted in a labeled ligase-ATP reaction intermediate that migrated close to the interface of the 1% and 4% gel gradient. In addition, nonspecific binding of ligase to the DNA resulted in slower migrating bands (lanes 2, 4 and 6) that disappeared in the presence of heparin, a commonly used competitor for non-specific binding of proteins to DNA (compare lanes 2–6 to 7–9). We added heparin at 20 μg/mL after the ligation reaction in all subsequent experiments to remove any non-specific binding of ligase to the DNA template. Visual inspection of the gel indi-cated that a significant fraction of the TECs acquired both the upstream and downstream DNA fragments during the ligation process (Fig. 3B).
TECs elongate efficiently on downstream DNA ligated to the scaffold. We next tested the ability of the TEC to elongate on the DNA ligated to the downstream end of the scaffold. To accomplish this, the DNA scaffold was designed as a U-less cassette and the first UMP base incorporated into the RNA
Figure 2. High efficiency in vitro reconstitution of active mammalian transcription complexes on nucleic acid scaffolds. (A) Features of the nucleic acid scaffold. The 14mer RNA is italicized and in bold letters. The 3'-end of the RNA that is complementary to the template strand oligo is shown in uppercase. (B) Schematic representation of experi-mental set up used to reconstitute TECs and to label the RNA in A15 TECs. A15 TECs were synthesized in the presence of α32P-ATP as a function of RNAP II concentration as described in “Experimental Procedures”. The A15 and the RNA species above it (since TECs had to first incorporate A15 to add the next base) were quantified, summed and plotted as a function of RNAP II concentration. (C) A20 TECs were synthesized in the presence of ATP and GTP and then elongated in the presence of ATP, CTP and GTP. The amount of run-off transcript (RO) synthesized relative to A20 TECs was quantified and is represented below the gel.
(Fig. 4B compare lanes 3 and 4, and Fig. 4C). We conclude this reconstitution system allows for effi-cient ligation of both upstream and downstream DNA, and the TECs assembled on this scaffold are able to elongate with high efficiency.
Transcript elongation by single molecules of RNAP II under assisting or hindering force in an optical trap. We used CT RNAP II assembled as A20 ECs to test our EC reconstitution/ligation substrates in the single-molecule assay. TECs containing CT RNAP II immobilized on beads resumed transcription in the presence of 1 mM NTPs. Example single-mole-cule transcription records obtained with assisting force DNA-pulling assay (Fig. 1A) are shown in Figure 5A. The average elongation rate of CT RNAP II under 10 pN of assisting force was ~10 nts-1, and transcrip-tion traces typically extended over several minutes. We observed pausing by individual molecules of CT RNAP II in the presence of either assisting (Fig. 5A) or hindering force (Fig. 5B). Examination of the elon-gation traces led to two important observations. CT RNAP II did not change average elongation velocity significantly either with assisting or hindering force, although we did observe some heterogeneity in elon-gation rate between individual traces. Similar rate
heterogeneity has been observed previously for bacterial RNAP, and ascribed to chemical het-erogeneity in the enzyme itself.16 Second, pausing can occur even with assisting force indicative of the propensity of RNAP II to enter inactive states even under conditions that favor forward trans-location. However, whether RNAP II enters such inactive states as a consequence of conforma-tional change due to the assisting force applied, or due to conformational change independent of it cannot be distinguished.
The efficiency of resuming transcript elonga-tion in the single-molecule laser trap assay using this template system was ~60–80% with an assisting force of 10 pN, and ~30% with 5 pN of hindering force. Hindering forces up to 5–7 pN were tolerated by the elongating RNAP II, although they frequently resulted in irreversible backtracking of the enzyme, or breakage of the
Figure 3. A tripartite ligation protocol to ligate DNA handles upstream and down-stream of TECs assembled on a nucleic acid scaffold. (A) Schematic representation of the tripartite ligation protocol. (B) Electrophoretic analysis of ligation to TECs on agarose gels. Reactions were fractionated on a step gradient agarose gel (top half at 1% and bottom half at 4%). Labeled TECs with scaffold, lane 1; upstream DNA ligated to TECs and with no heparin added, lane 2, or with heparin added after the reaction, lane 3; downstream DNA ligated to TECs and with no heparin added, lane 4, or with heparin added after the reaction, lane 5; upstream and downstream DNA ligated to TECs and with no heparin added, lane 6; or with increasing amounts of heparin added after the reaction, lanes 7–9. The position of the TECs, TECs ligated to upstream or downstream DNA, or both are indicated. The position of the reaction intermediate of ligase-NTP complex is also indicated.
Figure 4. TECs assembled on scaffolds elongate ef-ficiently on the ligated downstream DNA. (A) Struc-ture of the scaffold used to assemble TECs with halt position (A20) indicated. (B) Halted A20 TECs were elongated in the presence of 1 mM of indicated NTPs in the absence (lanes 1 and 2) or in the pres-ence (lanes 3 and 4) of upstream and downstream DNA ligated to the scaffold. The position of the halted complexes, elongation products and RO are indicated. The lanes enclosed by the box were used for quantification. (C) The lanes boxed in (B) were quantified and the fraction of the different RNA species plotted.
help stabilize the TEC via its interactions with the protein. In the present study we modified the scaffold further to increase the length of the template and non-template DNA strands to accom-modate formation of a halted complex. In addition, care was taken to design the sequence of the DNA strands to minimize the formation of any secondary structures that can potentially inhibit TEC reconstitution or elongation by RNAP, or both. In our defined reconstitution system, we achieved a reconstitution efficiency of ~80% using mammalian RNAP II. This efficient reconstitution system should be widely applicable to RNAPs from many types of organisms.
Elongation competency of the reconstituted TECs is critical for successful use of the system for analysis of transcript elon-gation in the single-molecule assay. The unavoidable physical loss of TECs and decrease in enzyme activity during subsequent manipulations required to observe transcription using the opti-cal trap dictates that the system be robust for reconstitution and elongation competency. The system we describe here supported
RPB1-antibody linkage used to immobilize the enzyme (data not shown).
We also tested the template system with yeast RNAP II immobilized using streptavidin coated poly-styrene beads via a biotin tag on the RPB3 subunit of the enzyme. Transcription traces were obtained for wild type yeast RNAP II at 1 mM NTPs with either assist-ing or hindering force in the absence of ammonium (Fig. 5C and D). We observed an elongation rate of ~25 nts-1 with assisting force, and ~23 nts-1 with hindering force. A detailed analysis of transcript elongation by wild-type and mutant yeast RNAP II is described in reference 27. Comparison of force-velocity curves for CT RNAP II and yeast RNAP II (Fig. 5E) led to three major observations. First, hindering force of 5 pN or assisting forces of up to 15 pN did not affect the pause free elongation rate by CT RNAP II, suggesting that the enzyme is not rate-limited for translocation. Second, CT RNAP II when transcribed in the presence of NH
4+ was
slower (by a factor of 4–5) than yeast RNAP II. Third, in the absence of NH
4+ yeast RNAP II was not limited
for translocation as the velocity was only marginally affected by increased assisting force. However in the presence of NH
4+, increased assisting force significantly
increased elongation rate by yeast RNAP II, indicating the enzyme becomes limited for translocation in the presence of NH
4+.
Discussion
We have developed an efficient template system that allows for high efficiency reconstitution of both yeast and mammalian RNAP II TECs. The salient features of the template system are (1) high efficiency TEC recon-stitution (~80%) of CT RNAP II; (2) highly active and efficiently elongating TECs upon the addition of NTPs (up to ~90%); (3) high-efficiency, tripartite ligation of long DNA tethers of choice upstream and downstream of the reconstituted TECs, allowing observation of many rounds of nucleotide addition and; (4) capacity to observe transcript elongation by either mammalian or yeast RNAP II using an opti-cal-trap assay under either assisting or hindering load.
In vitro TEC reconstitution was pioneered by Von-Hippel and coworkers,22 who established a system to assemble active TECs using E. coli RNAP and nucleic scaffolds containing tem-plate and non-template DNA strands, and a 12-nt RNA that was complementary to the template strand. Kashlev and coworkers refined this approach using an 8-nt RNA to reconstitute E. coli TECs,23 which was later modified to assemble TECs with yeast RNAP II and used to study transcription by single molecules of yeast RNAP II.19,20 We further modified this system to increase the length of the RNA to 14 nt, five bases of which at the 5'-end are non-complementary to the template strand DNA resulting in an 9-nt RNA:DNA hybrid, and established a high efficiency reconstitution system.24 The additional five bases at the 5'-end of the RNA will occupy the RNA exit channel of RNAP, and may
Figure 5. Transcript elongation by single-molecules of RNAP II in an optical laser trap. (A) Representative traces of transcript elongation by single-molecules of CT RNAP II over time at a constant assisting force of 10 pN. (B) Representative traces of transcript elongation by single-molecules of CT RNAP II over time at a constant hindering force of -5 pN. (C) Representative traces of transcript elongation by single-molecules of yeast RNAP II over time at a constant assisting force of 10 pN. (D) Representative traces of transcript elongation by single-molecules of yeast RNAP II over time at a constant hindering force of -5 pN. (E) Force-velocity curves of CT and yeast RNAP II. Velocity was measured in the presence or absence of 100 mM NH4
+, at 1 mM NTPs, and with assisting or hindering force. Values plotted are mean ± SEM.
interactions of RNAP II with nucleic acids in TECs and elonga-tion factors alter the force-velocity relationship. Thus, the tem-plate system we have established will allow for single-molecule studies to understand the elongation properties of mammalian RNAP II, the mechanisms that control it, and its regulation by elongation factors.
Experimental Procedures
Materials. All DNA and RNA oligonucleotides were from IDT and were gel purified by denaturing PAGE. FPLC-purified NTPs were from Promega Corp. α-32P-ATP was from Perkin-Elmer. T4 Polynucleotide kinase and T4 DNA Ligase were from NEB.
DNA. We used the 4.75 kb DNA fragment from the 5'-region of human Rpb1 gene encoding the UTR and sev-eral exons and introns to establish a template system to study transcript elongation by single molecules of eukaryotic RNAP II. The 4.75 kb fragment was PCR-amplified from HeLa cell genomic DNA and blunt-end cloned into pJET1/Blunt accord-ing to the manufacturers instructions (Fermentas). The result-ing plasmid, pPM172, was used as template to generate the 4.75 kb fragment for ligation downstream of the scaffold (see below). The fragment was PCR amplified using the forward primer 5'-CAC AGG CGC GCC CAC GGG GTG AGC AGT CAC G (#5975) that coded for a Dra III site (underscored) immediately upstream of the sequence of the hRPB1 gene, and the reverse primer 5'-TGC GGC GGG AAC ACA ACT GG (#5976) with a 5'-digoxigenin modification for the hindering load assay. The 2.7 kb DNA fragment to be ligated upstream of the scaffold was PCR amplified from the plasmid pRL702 using the forward primer 5'-CAG CGG TAA TTC CGA GCT GCA (#5,968) and the reverse primer 5'-CGA TTT CCA GCG CAC GTT TGT C (#5,969) with a 5'-digoxigenin modification for the assisting load assay. The upstream 2.7 kb and downstream 4.75 kb DNA fragments were restricted with Sty I and Dra III respectively and gel purified before use.
Proteins. Gdown1 free CT RNAP II was purified from calf thymus by PEI precipitation, anion-exchange chromatography on High-Q (Bio-Rad), 8WG16 antibody affinity chromatogra-phy and anion-exchange chromatography on Uno-Q (Bio-Rad) as described previously in reference 28. The purification of bioti-nylated yeast RNAP II is described in reference 27.
Scaffold assembly and TEC reconstitution. RNA and tem-plate DNA scaffold were assembled by annealing the template strand DNA (100 nM) oligonucleotide 5'-GTG GTG TCG CTT GGG TTC TCT TTT CGC CTT GTC GGC TGC GCG TCG GTG GGT GTT TCC TGA TGG CTG TTT GTT TCC TAT AGC (#5974) and the 14mer RNA (100 nM) 5'-UUU UUA CAG CCA UC (#3792) in RB (10 mM TRIS-HCl, pH 7.9, 40 mM KCl,) using a thermal cycler as described previously in reference 23. TECs were reconstituted in 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 of acety-lated bovine serum albumin/ml) by incubating either CT or yeast RNAP II (1–15 nM) with the scaffold (10 nM) containing tem-plate strand DNA and RNA at 30°C for 10 min followed by the
a very high elongation competency (~90%), making it possible to analyze a significant fraction of TECs using an optical-trap-ping assay. The high efficiency of elongation competency may be attributable to the reconstitution of the TEC with a GC rich RNA:DNA hybrid which could be further stabilized by the for-mation of a halted TEC containing 20 nt of nascent RNA.
Another feature that facilitated efficient use of the system in the single-molecule assay was the ability to ligate long fragments of DNA to the upstream and downstream ends of the TEC with high efficiency. Since we achieved very high efficiency of liga-tion, we could avoid use of restriction digestion to enrich for active TECs containing a downstream tether.19,21 Bypassing this step reduced the time spent by TECs at room temperature during restriction digestion, thus lowering the potential for TEC inac-tivation. It also avoided possible introduction of contaminating ribonucleases into the reaction. The absence of encoded Us in the DNA downstream of the site of TEC reconstitution allowed us to elongate the A20 TECs in the absence of UTP to position G76, which required the ligated downstream DNA that encodes U at position 77. High-efficiency ligation and the sequence at the ligation junction led to efficient elongation of TECs halted at position G76 on the ligated downstream DNA tether. By using DNA from the human RPB1 gene for the sequence of the downstream tether, we ensured that the behavior of RNAP II transcribing this DNA accurately reflects the protein-nucleic acid interactions that occur during native transcription rather than an artificial DNA sequence not normally encountered by the enzyme under study.
The template system we have established supported transcript elongation by a significant fraction of single molecules of CT RNAP II and yeast RNAP II subjected to assisting or opposing force in an optical laser trap. Despite the 4–5 times slower veloc-ity of CT RNAP II relative to yeast RNAP II, CT RNAP II was not significantly affected by modestly opposing or significantly assisting force (up to +15 pN; Fig. 5). Rather, the more rapidly elongating yeast RNAP II appeared more strongly affected by assisting force. This suggests that slower translocation was not the principle cause of slower transcript elongation by CT RNAP II. Given that the assays were conducted at high NTP concentra-tions (~1 mM), it is likely that CT RNAP II elongates more slowly because the catalytic step is slower than for yeast RNAP II (in the presence of 100 mM NH
4+). Although the pause-free velocity of
CT RNAP II (~9 nts-1) was not significantly affected by applica-tion of hindering or assisting force, the enzyme entered paused states even in the presence of assisting force. This observation might reflect the predisposition of CT RNAP II (or mammalian RNAP II in general) to readily enter non-productive pathways, and is consistent with the requirement of many elongation fac-tors for optimal transcription rates by mammalian RNAP II.1-4 These effects of force on mammalian RNAP II are preliminary and based on a limited number of observations. Thus, our con-clusions are tentative and will require more extensive data sets before strong conclusions can be drawn.
With more extensive data sets, however, the assay reported here will allow measurement of the force exerted by mamma-lian RNAP II during transcription and will allow study of how
Assay of transcript elongation by single molecules of RNAP II. Halted A20 TECs were made using either purified CT RNAP II or biotinylated yeast RNAP II and ligated to the 2.7 kb upstream and 4.5 kb downstream DNA fragments as described above. For the CT RNAP II single-molecule assay, 8WG16-coated polystyrene beads were washed and resuspended in PHC (50 mM HEPES, pH 8.0, 130 mM KCl, 5 mM MgCl
2, 0.1 mM EDTA,
0.1 mM DTT) plus 3 mg/mL BSA. This same procedure was used to prepare Streptavidin coated polystyrene beads for use in the yeast RNAP II single-molecule assay. In each case, the beads were sonicated for 5 min and incubated with approximately 2-fold excess A20 TECs (2 nM) for 30 min at room temperature, after which they were spun down and the supernatant discarded to remove NTPs and excess DNA handle left over from the ligation step. The pellet was resuspended in PHC, mixed with equimo-lar amounts of beads coated with anti-digoxigenin antibody, and incubated for another 30 min at room temperature. This two-bead mixture was diluted 30 fold in PHC containing an oxygen scaven-ger system13 and perfused into flow cells for use in the dual-beam optical-trapping microscope described previously in reference 5.
The distance between optically-trapped beads was converted into the DNA contour length5 and pauses were identified and scored as described previously in reference 29. Pause-free elonga-tion velocities of single RNAP II molecules were determined by fitting the velocity distribution to a sum of two Gaussians (one corresponding to pausing, and the other to active elongation) as previously described in reference 15.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
This work was funded by financial support from NIH/NIGMS (GM72795) to R.L., NIH/NIGMS (GM57035) to S.M. and D.O.D. (BC083495) and NIH/NIGMS (GM64474) to A.G.
Supplemental Materials
Supplemental material may be downloaded here: www.landes-bioscience.com/journals/transcription/article/20269
addition of non template strand (15 nM) oligonucleotide 5'-CAA GGC TAT AGG AAA CAA ACA GCC ATC AGG AAA CAC CCA CCG ACG CGC AGC CGA CAA GGC GAA AAG AGA ACC CAA GCG ACA CCA CGG G (#5973), and incubated for another 10 min at 30°C to complete the formation of TECs. The template and non-template DNA oligonucleotides were designed such that, upon annealing, will generate unique restriction sites for Sty I and Dra III at the upstream and downstream end of the scaffold, respectively.
Formation of labeled A20 TECs and ligation of upstream and downstream DNA to TEC. The RNA in the reconsti-tuted TECs was labeled by incubating the TECs with α-32P-ATP (10 μCi; 1 μM) for 2 min at 30°C to form A15 TEC. To make the halted A20 TECs used in all subsequent experiments, the labeled A15 TECs were incubated with ATP and GTP (100 μM each) for 5 min at 30°C. The 2.7 kb upstream and 4.75 kb downstream DNA fragments were then ligated to the A20 TECs by incubating the TECs (5 nM) with the DNA frag-ments (20 nM each) in EB in the presence of ligase (2K Units) and ATP (1 mM) at 12°C for 1 h. To monitor ligation of the fragments to the scaffold, ligations were initially performed in the absence of RNAP II. An aliquot of the reaction mixture was either separated on 1% agarose gel to visualize the appearance of a unique 7.5 kb band, or restricted with Kpn 1 and separated on a 4% agarose gel to visualize the appearance of a unique 1.22 kb band upon ligation of both upstream and downstream DNA fragments to the scaffold (Fig. S1). When labeled A20 TECs were used to ligate to the upstream and downstream DNA frag-ments, samples were incubated with heparin (20 μg/mL) for 5 min at 30°C after the ligation reaction to inhibit any non-specific interactions of the ligase to DNA. Aliquots were then separated on a step gradient agarose gel [top half of gel at 1% and bottom half at 4% (NuSieve)], and the wet gel exposed to a phosphor imager screen.
Transcript elongation by A20 TECs. After ligation of the DNA fragments to the labeled A20 TECs, the complexes were incubated with ATP, CTP and GTP (100 μM each) for 5 min at 30°C to form G76 TECs, which were then elongated in the pres-ence of all 4 NTPs (1 mM each) for 30 min at 30°C.
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