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Originally published In Press as doi:10.1074/jbc.M310290200 on November 6, 2003

J. Biol. Chem., Vol. 279, Issue 5, 3292-3299, January 30, 2004
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Diversity in the Rates of Transcript Elongation by Single RNA Polymerase Molecules*

Simon F. Tolic-Nørrelykke{ddagger}§, Anita M. Engh§||, Robert Landick**, and Jeff Gelles§{ddagger}{ddagger}

From the {ddagger}Niels Bohr Institute, Blegdamsvej 17, DK-2100 Copenhagen, Denmark, the §Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02454-9110, and the **Department of Bacteriology, University of Wisconsin, Madison, Wisconsin 53706

Received for publication, September 16, 2003 , and in revised form, November 5, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Single-molecule measurements of the activities of a variety of enzymes show that rates of catalysis may vary markedly between different molecules in putatively homogenous enzyme preparations. We measured the rate at which purified Escherichia coli RNA polymerase moves along a ~2650-bp DNA during transcript elongation in vitro at 0.5 mM nucleoside triphosphates. Individual molecules of a specifically biotinated RNA polymerase derivative were tagged with 199-nm diameter avidin-coated polystyrene beads; enzyme movement along a surface-linked DNA molecule was monitored by observing changes in bead Brownian motion by light microscopy. The DNA was derived from a naturally occurring transcription unit and was selected for the absence of regulatory sequences that induce lengthy pausing or termination of transcription. With rare exceptions, individual enzyme molecules moved at a constant velocity throughout the transcription reaction; the distribution of velocities across a population of 140 molecules was unimodal and was well fit by a Gaussian. However, the width of the Gaussian, {sigma} = 6.7 bp/s, was considerably larger than the precision of the velocity measurement (1 bp/s). The observations show that different transcription complexes have differences in catalytic rate (and thus differences in structure) that persist for thousands of catalytic turnovers. These differences may provide a parsimonious explanation for the complex transcription kinetics observed in bulk solution.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Different individual molecules within a purified enzyme or ribozyme preparation can display greatly differing rate constants for catalytic turnover (1–3). Some of this variability may be due to the presence of enzyme molecules with differing covalent structures; these may arise from the presence of two or more related genes, from alternative splicing of a single pre-mRNA, or from post-translational protein modifications (4). However, some heterogeneity may also arise from the adoption by enzyme molecules with identical covalent structures of different conformational states that persist through multiple catalytic turnovers (5, 6). It has been proposed that such conformational heterogeneity may be a heretofore unappreciated feature of macromolecular catalysts in general (2).

Bacterial RNA polymerases (RNAPs),1 and their structurally and mechanistically similar homologs, eukaryotic RNAP II enzymes, are large, multisubunit proteins that synthesize all cellular mRNAs (7, 8). These enzymes are processive; a given RNA molecule is synthesized in its entirety by the same RNAP molecule. In bacterial RNAPs, most RNA synthesis takes place in a stable transcription elongation complex (TEC) containing the core RNAP subunits, the template DNA, and the nascent RNA transcript. The TEC structure has been extensively characterized (9–12). A variety of proteins and DNA sequence elements in both prokaryotes and eukaryotes regulate gene expression by altering the extent to which active TECs terminate transcription upstream of or within genes (13–15). The efficiency of termination is strongly dependent on the rate of transcript elongation (16), which is modulated (and termination efficiency thus regulated) by accessory proteins that bind to the TEC as well as by sequence elements in the template or nascent transcript (17–20). Thus, full understanding of expression regulation requires knowledge of the mechanisms by which the rate of elongation is altered.

The mechanism of Escherichia coli RNAP transcript elongation has been investigated in vitro using macroscopic (i.e. performed on a molecular ensemble) kinetics experiments. These studies (17–19, 21, 22) have been interpreted as supporting complex mechanisms in which individual enzyme molecules can switch between two (or more) distinct, parallel reaction pathways by binding an allosteric regulator (17–19) or through conformational changes much slower than enzymatic turnover (23). However, uncertainty in the interpretation of macroscopic data can arise if the enzyme preparation contains molecular subpopulations with permanent differences in structure and kinetics.

In contrast, microscopic experiments that measure reaction rates of single enzyme molecules can clearly distinguish state switching in individual molecules from permanent heterogeneities in the molecular population (2). In such experiments on RNAP (24–29), each molecule is typically observed to move at a constant, single rate (except when pausing) over >1000 bp of template DNA. However, all such studies reported that different molecules in the population appear to move at different rates. The cause of this heterogeneity is not known, and previous reports do not present sufficient data to accurately characterize the width and shape of the velocity distribution.

To characterize the extent and causes of TEC kinetic heterogeneity, we investigated single-molecule elongation rates of specifically attached TECs at a well controlled temperature and zero applied force, conditions that are directly comparable with those of macroscopic studies. The experimental setup was optimized for high throughput, allowing us to collect comparatively large numbers of single-molecule data records and thereby accurately characterize the shape of the molecular population distribution of TEC elongation rates, not merely its average. The observed velocity distribution is unimodal to the limit of experimental error and is far wider than the variation in the reaction rates of individual TECs. The observations establish that even a highly purified preparation can contain multiple, kinetically distinct TEC species and raise concerns about the validity of interpreting macroscopic transcription kinetics using models that posit a homogeneous TEC population.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
The experimental procedures used by Yin et al. (25) to measure elongation rates of single TECs were modified to use a specifically bead-labeled RNAP derivative and improved procedures for data collection and analysis.

Chemicals—Nuclease-free bovine serum albumin (BSA; 20 mg/ml in 50 mM Tris-HCl, 100 mM NaCl, 0.25 mM EDTA, 1 mM 2-mercaptoethanol, 50% glycerol, pH 7.5) was from Roche Applied Science (catalog no. 711454).

Transcription Templates—DNA templates for transcription contained the strong promoter A1 from phage T7. 2651-bp linear double-stranded DNA labeled with digoxigenin at one end was produced by polymerase chain reaction amplification of pRL667 (26) with primers 5'-GTAAAACGACGGCCAGTG-3' and digoxigenin-3-OCH2(CONH(CH2)5)2-5'-pGAAACAGCTATGACCATG-3' (Integrated DNA Technologies, Coralville, IA). The transcribed sequence, a fragment of the E. coli rpoB gene, contains no known factor-independent terminators or strong regulatory pause sequences.

Beads—Avidin-coated, 199 ± 0.3 (S.D.)-nm diameter polystyrene microspheres, "beads" (Seradyn Inc., Indianapolis, IN), were prepared as in Yin et al. (25). BSA was added to the final bead preparation to a concentration of 5 mg/ml.

Biotinated RNAP—Biotinated RNAP was obtained from the combined cells from two 10-liter cultures of E. coli K12 strain RL674. Cultures were grown to late log phase in a New Brunswick fermenter in 2x LB medium (30) supplemented with 2.5 µg of biotin/ml. The enzyme was purified as described by Hager et al. (31) and stored at –80 °C in 50% glycerol, 10 mM Tris-HCl, pH 8.0, 0.1 mM Na2EDTA, 100 mM NaCl, 1 mM MgCl2. RL674 (W3110 F– {lambda}– tnaA2 trpR rpoC3530[{beta}'::BCCP] zja::kan) was prepared by P1 phage transduction (30) of an rpoC::fabE' fusion from strain RL670 (RL670 is strain JC7623 carrying rpoC3530({beta}'::BCCP) zja::kan) into strain CY15001 (32) by selecting for kanamycin resistance encoded by the zja::kan marker that is tightly linked to rpoC3530. RL670 was obtained by selecting for double crossover of the rpoC3530 zja::kan region from pRL648 in a pRL648 transformant of JC7623 (33) using 25 µg of kanamycin/ml in LB agar plates as described by Oden et al. (34). pRL648 was derived from the rpoC-carrying plasmid pRW207 in the following steps: (i) introduction of XhoI and StuI sites at the C terminus of rpoC by oligonucleotide-directed mutagenesis of a single-stranded form of pRW207 to yield pRL622 (oligonucleotide: 5'-TACGTTATTTGAGGCCTAACTCGAGCTCGTTATCAGAACCGCCCA-3'); (ii) ligation of a 288-bp XhoI, StuI-digested PCR fragment containing the C-terminal 86 codons of E. coli fabE from chromosomal DNA of strain CY15001 between the XhoI and StuI sites of pRL622 to yield pRL625 (PCR oligonucleotides: 5'-CACCTCGAGGCGCCAGCAGCAGCGGAAATCA-3' and 5'-CACAGGCCTCAATTTTATCCAGCATCTTCG-3'); (iii) ligation into the HindIII site of pRL625 of a 1374-bp HindIII-Eco47III fragment carrying kan from the E. coli transposon Tn5 (proximal to rpoC; obtained from the plasmid pKIXX from Amersham Biosciences) and a 2326-bp SspI-HindIII fragment carrying the region immediately downstream of rpoC from pNF1346 (distal to rpoC (35)) to yield pRL648. pRW207 is identical to pRW308 (36) except that it contains a wild-type lacI gene and a wild-type rpoC translation initiation signal.

Stalled TECs—Transcription elongation complexes stalled at position +20 (310 bp from one end of the DNA) were prepared and purified as described by Yin et al. (25). The fraction of TECs capable of producing a full-length 2342-nucleotide run-off transcript, assayed as described by Landick et al. (37) with minor modifications, was ~80%.

Preparation of Samples for Microscopy—Microscopy flow cells (volume ~30 µl) were prepared as described (25), except that the spacers were a single thickness of Parafilm (American National Can, Menasha, WI) coated with silicone vacuum grease (Dow-Corning). Before use, the working surface was rinsed with ethanol, dried with pressurized air, rinsed with autoclaved water, and again dried. Stalled biotinated TECs were mixed with avidin-coated beads and incubated ~15 h at 4 °C. Anti-digoxigenin was attached to the cell surface as described by Finzi and Gelles (38) except that the incubation was ~15 h at 4 °C. The cell was then washed with 300 µl of PTC-SB buffer (20 mM Tris-Cl, pH 8.0, 130 mM KCl, 4 mM MgCl2, 0.1 mM Na2EDTA, 0.1 mM dithiothreitol, 20 µg/ml acetylated BSA, 80 µg/ml heparin) and incubated with 2.0 mg/ml BSA in PTC-SB for 5 min to saturate the surface with protein in order to minimize nonspecific binding of beads. The bead-TEC mixture was then introduced and allowed to bind for 60–120 min at 24 °C; unbound beads and complexes were removed by washing with 300 µl of 0.5 mg/ml BSA in PTC-SB.

Activity of Stalled Complexes—To restart transcription, ribonucleoside triphosphates were introduced by flowing into the cell 100 µl of 0.5 mM each ATP, GTP, CTP, and UTP in PTC-SB supplemented with 0.5 mg/ml BSA. TPM experiments revealed that 140 of 341 total bead tethers observed shortened upon the addition of NTPs; thus, 41 ± 6% of the immobilized, bead-labeled transcription complexes are active. In control experiments in which beads were presaturated with biotin before mixing them with stalled TECs (to block the specific interaction between the biotinated polymerase and the avidin-coated beads), the fraction active was reduced by 56%, and the surface density of tethered beads was reduced by 50% relative to comparable samples without added free biotin. We therefore estimate an upper limit of 22% (equal to (1–0.50) x (1–0.56)) on the fraction of active complexes observed in the experimental samples that result from purely nonspecific attachment of biotinated RNAP to the beads. The number of nonspecific attachments is likely to be smaller than this estimate, since the number of nonspecific attachments is expected to be higher in the control experiments due to the higher concentration of unbound complexes. However, there remains a formal possibility that additional nonspecific interactions might occur in TECs already specifically bound to beads.

Temperature Control—Transcription reaction temperature was controlled by holding the microscope room at a constant temperature. The temperature at the start of each reaction was 24.0 ± 0.4 °C (mean ± S.D.; range 23.5–24.9). Measured reaction rates displayed a weak linear correlation with the temperature of slope 3.7 bp s–1 (°C)–1. Thus, the predicted variation in rate due to temperature differences between reaction runs is small (1.4 bp s–1 root mean square) and was therefore neglected in subsequent analysis.

Data Collection and Image Processing—Observation of the tethered beads by video-enhanced differential interference contrast light microscopy was as previously described (24). Tether length (L) was determined as described by Yin et al. (25), except that the Brownian motion was calculated as {delta}' = ST – SS, where ST and SS are the image size parameters of a tethered and an immobile bead in the same microscope image. Minor differences in the diameters of different beads are not expected to cause significant error in L, since the calibration factor (25) depends only weakly on bead diameter (<28% change over a diameter range of 98–230 nm) (25).2

Transcription Rates—Transcription rates for single TECs were determined by linear fitting of tether length records. Fits minimized the following,

(Eq. 1)
where n is the number of data points in the record, Li are the measured tether lengths, {Lambda}i are the fit tether lengths ({Lambda}i = Ati + B, where ti are the times and A and B are the rate and initial length fit parameters, respectively), and {pi}i = 0.092{Lambda}i + 9.7 bp are the iteratively determined precisions (25) of each data point. Some records contained rare transient excursions such as those caused by brief sticking of the tethered bead to the surface. To minimize the effect of such events, data points that deviated from the fit line by >2{pi}i were iteratively excluded from the fit; typically, <=1 such point was excluded per record (mean record length was 22 points (95 s)). The backing of the individual linear fits, determined from the {chi}2 value, was typically >95%.

The histogram of transcription rates was fit with a Gaussian after iterative exclusion of measurements (3 of 140 total) with rates above a threshold (r) three S.D. values from the mean value (Chauvenet's criterion (39)). The fit minimized the following,

(Eq. 2)
subject to the constraint,

(Eq. 3)
where m is the number of fitted histogram bins below r, Cj is the observed number of events in the jth bin, g(x) is a Gaussian, and Gj is the area under the Gaussian fit in the interval covered by the bin. When necessary, bins in the histogram tails were combined so that the following was true,

(Eq. 4)
where xj and xj + 1 were the lower and upper bin limits, respectively.

Extent of RNAP Biotination—To measure the extent of RNAP biotination, core polymerase subunits ({beta}'{beta}{alpha}2 without {sigma}) were formed into transcription complexes at a final concentration of 2 µM using the method of Siderenkov et al. (40). Transcription complexes were formed in 10 mM Tris-HCl, 40 mM KCl, 5 mM MgCl2 with equimolar concentrations of polymerase, DNA oligonucleotides that formed a synthetic transcription bubble of 12 nucleotides (template strand, 5'-CTCTGAATCTCTTCCAGCACACATTAGGACGTACTGACC-3'; nontemplate strand with mismatched region underlined, 5'-GGTCAGTACGTCCATTCGATCTCCCGAAGAGATTCAGAG-3'; obtained as gel-purified oligonucleotides; Genelink, Inc., Hawthorne, NY), and an RNA oligonucleotide complementary to the unpaired portion of the template DNA strand (5'-AUGUGUGCU-3'; Dharmacon, Inc., Lafayette, CO). The transcription complexes were diluted (200 nM final concentration) into buffer (10 mM Tris-HCl, 40 mM KCl, 150 mM NaCl, 5 mM MgCl2, 5% glycerol) with or without 10 µM streptavidin, incubated for 10 min at 24 °C, and then loaded on a 4% NuSieveTM agarose electrophoresis gel (FMC, Inc., Rockland, ME). Streptavidin was bound by the biotinated RNAP and retarded the electrophoretic mobility of the corresponding transcription complexes but had no effect on the electrophoretic mobility of transcription complexes containing wild-type RNAP. A small, variable fraction of the transcription complexes containing biotinated RNAP were observed to self-associate under the low ionic strength electrophoresis conditions, yielding a slower mobility band that was independent of the presence of streptavidin. Self-association of wild-type RNAP at low salt has been previously reported (41, 42).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Rationale—In previous studies, we observed that individual RNAP molecules almost always moved along template DNA at a rate that was constant to the limit of experimental uncertainty. However, the transcription rates of individual molecules were not identical. Instead, different molecules moved at different rates, the fastest having a velocity >5-fold larger than the slowest. Yin et al. (25) first demonstrated that this variability in kinetic properties from one individual TEC to the next was considerably larger than the experimental uncertainty in the rate measurement.

Some or all of the previously observed variability might be an artifact of the methods used to measure single-molecule elongation rates. Sustained observation of TECs by single-molecule light microscopy techniques requires immobilizing the complexes to prevent them from diffusing out of the field of view. In our previous studies, TECs were immobilized by nonspecific adsorption to a glass surface (24–26, 43, 44). Surface adsorption has been demonstrated to alter the activity or conformation of a variety of enzymes and proteins (45, 46) and thus might cause different RNAP molecules to elongate transcripts at different rates. Additionally, an external mechanical load was applied between the RNAP and the downstream end of the template in many previous studies (27–29, 43, 44, 47). Since the orientation of the applied force with respect to the enzyme may be different in different molecules, force application could also introduce kinetic heterogeneity in the molecular population, even at forces too low to have a measurable effect on the population-averaged velocity. Furthermore, the laser traps used to apply force can cause local heating (48), resulting in further variability in reaction rates. To eliminate these possible artifactual causes of molecular heterogeneity, we made new measurements of the elongation rates of single molecules of an RNAP derivative attached through a specific biotin-avidin linkage; rates were measured using the zero applied force tethered particle motion (TPM) method.

Improved Method for Measuring the Distribution of Single-molecule Transcript Elongation Rates—A polymerase derivative bearing a single biotin moiety was expressed using a recombinant gene encoding 86 amino acids from the C terminus of the E. coli biotin carboxyl carrier protein subunit (BCCP) of acetyl-CoA carboxylase fused (through a single Leu residue) to the C terminus of E. coli RNAP {beta}' subunit. This construct is expected to be post-translationally biotinated in vivo at a Lys residue 35 amino acids from the C terminus (49). A strain containing no other gene encoding {beta}' aside from the recombinant gene is viable, demonstrating that the fusion retains biological activity. The highly purified (31) polymerase preparation from this strain (Fig. 1A) exhibits a transcript elongation rate similar to that of wild-type enzyme in bulk assays (50).3 In native gel electrophoresis, the mobility of monomer TECs made from this preparation is efficiently shifted in the presence of excess streptavidin (Fig. 1B). This demonstrates that essentially all of these TECs are biotin-labeled.



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  		FIG. 1.
The biotinated RNAP derivative and the design of the single-molecule transcript elongation experiment. A, denaturing SDS-polyacrylamide gel electrophoresis (Coomassie Blue R stain) of wild-type (WT) RNAP holoenzyme (left) and the biotinated (BIO) derivative (right). {beta}'-BCCP is a fusion of the RNAP {beta}' subunit with the E. coli biotin carboxyl carrier protein. B, native gel electrophoresis of transcript elongation complexes in the presence or absence of streptavidin. 200 nM core RNAP (biotinated in lanes 1 and 2 and wild type in lanes 3 and 4) was formed into ternary transcription complexes by binding to a scaffold consisting of bubble duplex DNA oligonucleotides paired to a 9-bp RNA (see "Experimental Procedures"). The mixture was then incubated with or without 10 µM streptavidin (SA) and electrophoresed through a 4% agarose gel, which was stained with ethidium bromide and photographed on a UV transilluminator (wavelength 302 nm). Schematic diagrams (top to bottom) represent streptavidin-bound transcription complexes, unbound complexes, and scaffold alone. The minor low mobility bands are assumed to arise from self-association of RNAP at low salt (see "Experimental Procedures"). C, schematic diagram (not to scale) of the single-molecule transcript elongation experiment (see description under "Results").

 

To measure elongation rates of single, specifically immobilized TECs, we prepared microscope samples in which biotinated TECs labeled with avidin-coated 199-nm diameter polystyrene beads were linked to an anti-digoxigenin-coated glass coverslip through a digoxigenin moiety incorporated at the downstream end of the DNA template (Fig. 1C). TECs stalled by withholding UTP were prepared from the biotinated polymerase and a 2651-bp digoxigenin-labeled template DNA. Then the bead-labeled TECs were introduced to a coverslip flow chamber pretreated with the antibody. This protocol allowed reproducible preparation of samples with typically ~10 surface-tethered, bead-labeled TECs per video microscope field (1240 µm2), of which roughly half could be reactivated upon the addition of all four NTPs. Movement of the reactivated TECs along the template DNA was measured by TPM methods previously employed to study the movements of RNAP (24) and RecBCD (51) along DNA. These methods measure the extent of Brownian motion of a DNA-tethered bead as a function of time. Using an experimentally determined relationship between the Brownian motion and the tether length, the latter is derived, and thus the position of the enzyme on the DNA is measured. TPM methods have the ability to simultaneously observe movement from multiple TECs in a single sample and thus allow comparison of single-molecule recordings collected under precisely identical ambient conditions. Even more importantly, this experimental design allows more efficient collection of single-molecule transcription data relative to that from laser tweezers experiments, which observe only one TEC at a time. Thus, the design facilitates obtaining data records in the quantities necessary to accurately characterize the width and shape of the distribution of rates across the molecular population.

Single-molecule Transcript Elongation Rates—A total of 140 single-molecule elongation events were observed; example records are shown in Fig. 2. The vast majority (128 of 140 records) showed that transcription velocity was constant to the limit of experimental error. However, rare examples of pausing (n = 7) and abrupt changes in velocity (n = 7) were observed (e.g. see Fig. 3).



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  		FIG. 2.
Records of 13 transcription complexes active simultaneously in the same microscope field at 0.5 mM each ATP, GTP, CTP, and UTP at 24.5 °C. Single-molecule elongation rates ± S.E. were determined as the slopes of linear fits (solid lines) and ranged from 10 to 42 bp/s. The average rate in these records was 20 ± 7 (S.D.) bp/s.

 



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  		FIG. 3.
An unusual lengthy pause in transcript elongation by a single TEC. This complex transcribed at a rate of 17.6 ± 3.0 (S.E.) bp/s, paused (rate 0.81 ± 1.90 bp/s) for ~55 s, and then resumed transcription at the initial rate of 17.7 ± 1.1 bp/s. The temperature was 24.0 °C.

 
The records shown in Fig. 2 were collected simultaneously from the same microscope field. They illustrate that single-molecule elongation rates measured under identical conditions of temperature, pH, NTP concentrations, and buffer composition show a substantial variation in elongation rates. All complexes transcribed at a constant velocity throughout the reaction, yet the velocities of individual complexes differed greatly, ranging from 10 to 42 bp/s.

The majority of data records (103 out of 140) were collected from microscope fields that had more than two active complexes. The width of the velocity distribution in each of these individual microscope fields is 6.8 ± 0.8 bp/s (root mean variance ± S.E.; n = 33 fields), whereas the width for all 140 records taken as a single group is 7.7 bp/s (S.D.). Thus, the spread in elongation rates in individual microscope fields is comparable with the spread in elongation rates for the entire population, suggesting that little or none of the width of the population rate distribution is caused by variation in experimental conditions from one microscope field to another.

A small subpopulation of the beads observed in our experiments might be linked to the surface through more than one active transcription complex. TPM measurements on such beads would in general overestimate single-molecule transcription rates, since geometry dictates that the amplitude of Brownian motion will decrease faster if a bead is attached to two spatially separated enzyme molecules than if it is linked to only one. To determine whether the presence of a subpopulation of multiply tethered beads contributes to the observed variability in rate, we conducted experiments in which stalled TECs were mixed with beads at two different molar ratios, 1:1 or 1:10. At the latter ratio, no more than 5% of beads will have two or more TECs, assuming independent binding. The fraction of beads in which multiple tethering affects the measured rate will be substantially lower than this upper limit, since not all TECs are active and not all bind to beads. The velocity distribution observed at the higher 1:1 ratio is essentially identical to that at 1:10 (Fig. 4), demonstrating that there is no significant multiple tether effect on the velocities even at the higher ratio. In independent optical trapping experiments, beads tethered by TECs made from the same biotinated RNAP preparation display the persistence length characteristic of single duplex DNA molecules (47).



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  		FIG. 4.
Histogram (bin width 2.16 bp/s) of transcription rates for 140 individual transcription complexes. A fraction of records in each bin was recorded at a 1:10 molar ratio of TECs to beads (dark gray); the remainder was at 1:1. A Gaussian fit to the aggregated data (solid line) yields a mean rate of 15.9 ± 0.6 (S.E.) bp/s and root mean square distribution width of 6.7 bp/s. Dashed lines show the confidence interval (± one S.D. value) on the count number predicted by the fit. Three outlier measurements more than three S.D. values from the mean (asterisks) were not included in the fit.

 
Distribution of Transcription Rates—Data from both single molecule and bulk experiments have been interpreted as evidence for the existence of two (or more) subpopulations of TECs (23, 52) that move at rates differing by a factor of ~2-fold (27) or more (22). The comparatively large number of single-molecule elongation rates measured here provides a clear picture of the population distribution of rates (Fig. 4). The distribution is unimodal; there is no evidence for a small number of discrete populations with greatly differing transcription rates. Rather, TECs appear to exist in a variety of velocity states that are distributed relatively uniformly about a single mean.

Some of the width of the observed rate distribution (Fig. 4) is expected to arise merely from random errors in the experimental determination of the velocities of individual molecules. Since single-molecule velocities were measured as the slope of a line fit to the position versus time records, the S.E. on the slope determined by the weighted least-squares fit is an unbiased estimator of the random error in the velocity measurement. This velocity error tended to be larger for faster moving complexes (data not shown); this result is expected, since these complexes had shorter duration position records and thus less data available for fitting. The average of the velocity error over the set of 137 data records was 1.3 ± 0.9 (S.D.) bp/s (range, 0.11–5.1 bp/s). Thus, the observed width of the single molecule transcription velocity distribution (6.7 bp/s; Fig. 4) is more than 5 times broader than that expected from experimental error alone.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Time Invariance of Single-molecule Transcription Rates
Davenport et al. (27) reported that individual transcript elongation complexes are observed to switch between quickly and slowly elongating states. Translocation velocities of the two states varied by ~2-fold, and molecules were observed to remain in one state for average periods of ~6 min (~1500 bp) before switching. In contrast, other recent studies observed no velocity switching by single molecules of wild-type E. coli RNAP (29, 47), although rare switching was detected with the RpoB8 mutant protein (29). In the large number of single molecule records collected for the present study, events consistent with a discrete >=2-fold change in velocity were observed only so infrequently (<4% of records) that artifacts (e.g. more than one TEC linking the bead to the surface) cannot be excluded as the cause. In addition, the population distribution of single-molecule velocities (Fig. 4) shows only a single, broad peak. Thus, whereas the data suggest the presence of >>2 species of TEC, each with its own characteristic elongation rate, they show that true velocity state switching is either highly infrequent (less than once per 104 bp transcribed) or nonexistent under the conditions we used. These conclusions do not necessarily contradict those from previous observations of velocity state switching, because those were made under different conditions (200 µM NTPs; 20 °C (27)) or with a mutant RNAP (29). Indeed, the two states proposed by Davenport et al. (27) might be a subset of the larger number of rarely interconverting or noninterconverting molecular states required to explain the observations reported here.

Elimination of Artifactual Sources of Variation in Measured Single-molecule Transcription Rates
The experimental methods used to measure single-molecule transcription velocities might in principle induce molecule-to-molecule variation in the elongation rate measurements. Before concluding that the observed heterogeneity in the rates is a property inherent to the TECs themselves, it is prudent to consider the extent of variability caused by a variety of possible experimental artifacts.

Temperature Variation—Since all single-molecule records were not recorded simultaneously, inadequate temperature control could lead to variation in rate between different records. In the experiments reported here, the temperature was kept at 24.0 ± 0.4 °C for all recordings. Temperature changes over this range have an insignificant effect on transcription rate (see "Experimental Procedures"). In addition, even TECs recorded simultaneously in the same microscope field, and thus at the same temperature, displayed the full range of observed velocities (Fig. 2).

Variability in the Local Concentrations of Solutes—The rate of transcription is dependent on the concentrations of solutes such as NTPs and Mg2+. If the concentrations of such species differed from one experiment to the next, this would widen the observed rate distribution. However, changes in solute concentration cannot alone account for the range of single-molecule rates observed, since wide variations in rate are seen even within a single microscope field in one sample (Fig. 2). The root mean square time for an NTP molecule to diffuse across the ~30-µm-wide microscope field is ~1.5 s (diffusion coefficient 3 x 10–5 cm2/s) (53); therefore, any local concentration gradients across the field that might exist when NTPs are first introduced to the sample are unlikely to persist long enough to affect the measured rates.

DNA Configurational Entropy—As transcription proceeds, the DNA both upstream and downstream of the polymerase becomes more restricted in its conformation as the bead approaches the surface. The polymerase must therefore do work against the configurational entropy of the DNA and the rotational and translational entropy of the bead. However, even in an extreme model (see Supplemental Material) in which the DNA and bead are completely immobilized when the bead reaches the surface, the force from entropic elasticity acting against polymerase movement is no larger than 0.1 pN. Since this upper limit on the entropic force is far less than the forces required to significantly alter the time-averaged DNA configuration, the entropic force is unlikely to introduce heterogeneity in single-molecule elongation rates even if the time-averaged direction of the force with respect to the orientation of the complex differs between individual complexes.

DNA Topological Heterogeneity—The contour length of the DNA template used in these studies is similar to the circumference of the 199-nm diameter beads. Consequently, it is possible that in some complexes the DNA might be trapped in a knotted configuration when it is attached to the surface or while the tether is shortening. However, the probability of trapping a spontaneous knot in a DNA of the length used here is <<2% (54). Thus, knotting in the tethered elongation complexes is unlikely to contribute significantly to the observed width of the velocity distribution.

Nonspecific Binding of Transcription Complexes—In many single-molecule studies of RNAP transcript elongation, TECs were immobilized by nonspecific adsorption to a surface (24–26, 43, 44, 55, 56). The mean elongation rate measured in most of those studies corresponded closely to that determined in bulk experiments on TECs free in solution. Despite the similar average rates, it is possible that uncontrolled adsorption might induce changes in TEC structures or dynamics. Such changes might cause populations of immobilized complexes to display additional kinetic heterogeneity over that of populations of complexes in solution. In more recent work, nonspecific attachment has been replaced by specific linkage of a modified RNAP derivative to surface-attached streptavidin, avidin, or antibody molecules (this study and Refs. 27, 28, 29, 47). In these studies, adsorption-induced changes in TEC structure should be eliminated, and TEC orientation with respect to the surface normal should be considerably more uniform. However, the velocity distribution measured in our experiments (in which at most a small subpopulation of complexes are not specifically bound; see "Experimental Procedures") is not significantly narrower than those in the previous studies in which all complexes are nonspecifically bound. Therefore, it is unlikely that nonspecific binding causes substantial kinetic heterogeneity in this or earlier studies.

Intrinsic Heterogeneity in Transcription Elongation Complexes
In the work presented here, substantial rate variation between different individual transcription elongation complexes is observed, although the experiments are designed to minimize the major possible artifactual causes of such heterogeneity. Such heterogeneity was also seen in previous single-molecule studies of transcript elongation (25–29, 44, 47), with population S.D. values 18–114% of the mean velocity ((6.7 bp/s)/(15.9 bp/s) = 42% in the present study; 40, 67, 38–47, 55–114, 18, 30, and 52%, respectively, in the studies cited above (a range is given when more than one concentration of NTPs was used)). Thus, significant kinetic variation across a population of transcription complexes is not restricted to the RNAP preparation or experimental protocol used here. Interestingly, the observed variation in velocity is of similar magnitude to the variation in transcriptional stall forces measured in optical trapping experiments (43, 44, 47). Although the mechanism of force-induced stalling is not fully understood, the nonlinearity of the TEC force-velocity curve suggests that different reaction steps are rate-limiting at low and high loads. This implies that zero-force velocity and stall force depend on the rate constants of two (or more) different reaction steps (43). Thus, it is unlikely that the observed heterogeneity in the molecular population is confined to a single step in the enzyme mechanism.

A recent optical trapping study (29) examined a set of single-molecule transcript elongation records and stated that this population of wild-type TECs was found to be homogeneous. However, the data supporting this conclusion were not presented, and no estimate was given of the level of heterogeneity that would have been detectable given the comparatively small size (30 records) of the data set. Significantly, the same report does describe substantial molecule-to-molecule variability with respect to the time spent in short transcriptional pauses, and the reported width of the distribution of time-averaged single-molecule velocities (2.1 bp/s root mean square variation on a mean of 12.0 bp/s), which include these pauses, is roughly consistent with the magnitude of heterogeneity measured here and in the earlier single-molecule studies.

Molecular Heterogeneity and Macroscopic (Bulk) Kinetics
Heterogeneity in populations of transcription complexes is consistent with the complex kinetics previously reported for steady-state and pre-steady-state transcript elongation in macroscopic experiments on E. coli RNAP and Saccharomyces cerevisiae RNAP III (21, 22, 57). In those reports, heterogeneous elongation kinetics were rationalized by assuming that a homogeneous population of transcription complexes partitions between two (or more) parallel kinetic pathways during transcript elongation. However, some of the observed behavior may also be explained by the existence of multiple subpopulations of transcription complexes with permanent differences in their functional properties. The single-molecule experiments reported here provide a direct test of these alternative explanations for the behavior seen in bulk and demonstrate that different individual transcription complexes move at different speeds that are constant within experimental uncertainty over thousands of catalytic turnovers.

An alternative, macroscopic test for kinetic heterogeneity in TEC populations was performed by Pasman and von Hippel (58, 59), who measured termination efficiencies on a transcription template containing tandem repeats of identical terminators. Successive terminators were found to have similar termination efficiencies, consistent with the interpretation that the population of TECs is kinetically homogenous (at least with respect to termination propensity). However, heterogeneity in elongation velocities of the magnitude reported here would probably produce only small changes in termination efficiency in this experiment. In particular, the change in termination efficiency at successive terminators is predicted to be less than the reported variability in the termination efficiency measurements if direct kinetic competition between elongation and termination is assumed (see Supplemental Material).

Possible Structural Origins of TEC Kinetic Heterogeneity
TECs that elongate RNA at different rates must have different three-dimensional structures. Since the distribution of population velocities (Fig. 4) is smooth and ~5 times wider than expected for a single species moving at constant velocity, it is likely that contributions from three or more distinct species of transcription complexes each with different elongation velocities are required to explain its shape. The differences in TEC structures that account for their differing kinetics are unknown. One hypothesis is that kinetic heterogeneity might arise from stable differences in the way the protein binds to template, transcript, or some allosteric regulator. The last possibility is intriguing because of reports of allosteric regulation of RNAP by nucleoside triphosphates (22, 60). However, the reported nucleoside triphosphate regulation of elongation appears to saturate at ~5 µM NTPs and thus is unlikely to account for the heterogeneity observed here at 0.5 mM NTPs. Allosteric regulation of E. coli RNAP by specific transcript sequences is well established (19, 61, 62), but the transcription template used here encodes no known regulatory sequences. On the other hand, the origin of kinetic differences between molecules might be purely conformational; it has been speculated that RNAP can adopt a large number of long-lived conformational substates with differing catalytic properties (23). However, direct experimental evidence for purely conformational heterogeneity is lacking.

An alternative hypothesis is that molecule-to-molecule kinetic variations arise from the presence of RNAP molecules that differ in covalent structure. Although the RNAP preparation used here is purified by high resolution ion-exchange chromatography from a strain containing single copies of the core polymerase structural genes, it may nevertheless contain unresolved subpopulations of protein molecules with different primary sequences or post-translational modifications. Protein synthesis error rates in E. coli are reported to be between 3 and 20 misincorporations per 104 residues (63, 64); this would yield on average one or more amino acid substitutions in each RNAP molecule and might explain some of the observed differences in catalytic properties. Perhaps more significantly, E. coli RNAP may be subject to enzymatic or nonenzymatic post-translational modification. Preparations purified from cultures at different growth phases show differences in promoter recognition arising from changes in the relative amounts of at least three chromatographically separable forms of the core enzyme, consistent with the existence of post-translational modifications that modulate enzyme activity (65). Further work is required to determine whether the heterogeneity in single molecule velocities arises from such modifications.

Effect of Transcriptional Pausing on Overall Elongation Rate
Optical trapping studies of single TECs at the high spatiotemporal resolution achievable at high applied forces demonstrate that forward motion is frequently interrupted by one or more classes of short duration (on the order of 1–10 s) pauses (29, 47). Molecule-to-molecule variation in the time-averaged elongation velocity may therefore arise from differences in the velocity between pauses, the frequency of pauses, the duration of pauses, or some combination of these factors. Adelman et al. (29) analyzed optical trapping records assuming a two-state run-pause model and concluded that the rate differences between different molecules could be attributed mainly to variability in the fraction of time spent in the paused state(s). In contrast, Neuman et al. (47) detected substantial heterogeneity both in the kinetic properties of pauses and in the elongation velocity between detected pauses and demonstrated the presence of at least two distinct species of paused TEC. The TPM method used here does not resolve short pauses; thus, our data do not address the discrepancies between the two studies. Nevertheless, the shape of the population distribution (Fig. 4) suggests strongly that there are multiple, kinetically distinct species of TEC. This conclusion is independent of any assumption of a specific run-pause state model. Furthermore, the population variability observed in our experiments at zero applied force is approximately the same as or greater than that seen in the optical trapping studies at high forces. Thus, there is no evidence that the application of force increases the observed population heterogeneity of the time-averaged elongation rates.

Do All Enzymes Display Substantial Molecule-to-molecule Variation in Catalytic Rate?
Population variability of a magnitude similar to that seen with E. coli RNAP has been detected in single molecule studies of a number of other protein and RNA enzymes, including DNA polymerase (66, 67), cholesterol oxidase (2), lactate dehydrogenase (1), alkaline phosphatase (4), hairpin ribozyme (3, 68), and RecBCD (51, 71).4 These observations have led to suggestions that such variability is an inevitable feature of enzymatic catalysis, perhaps arising from a "rough energy landscape" of macromolecular conformational substates. However, other enzymes display no significant molecule-to-molecule variation in catalytic rate. Kinesin (69) and E. coli topoisomerase II (70) are two examples of enzymes that have been studied extensively in single molecule experiments and display highly uniform catalytic rates across the molecular population. Thus, such variation is neither an inevitable artifact of single molecule measurements nor a universal feature of enzymatic catalysis. Definitive conclusions about the prevalence and significance of molecule-to-molecule kinetic heterogeneity will require further investigation into its causes.

Conclusions
With rare exceptions, individual RNAP molecules were observed to move on DNA at a constant characteristic velocity. However, these characteristic velocities are different for different molecules, displaying a broad, unimodal distribution across the molecular population. We hypothesize that the kinetic heterogeneity arises from the presence of TECs with multiple structures. Heterogeneous post-translational modification of RNAP is one possible source of such structural differences. The presence of multiple stable species of TEC may suggest alternative interpretations for data from a number of existing bulk (ensemble average) studies of TEC structure and mechanism. Elucidation of the structural origins of the observed kinetic heterogeneity would aid in assessing its mechanistic consequences and biological significance.


   FOOTNOTES
 
* This work was supported by grants from NIGMS, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

The on-line version of this article (available at http://www.jbc.org) contains two additional figures. Back

Present address: LENS, Polo Scientifico, Via Nello Carrara 1, 50019 Sesto Fiorentino, Florence, Italy. Back

|| Present address: Dept. of Molecular and Cellular Physiology, Stanford University, Stanford, CA 94305. Back

{ddagger}{ddagger} To whom correspondence should be addressed: Dept. of Biochemistry, MS009, Brandeis University, P.O. Box 549110, Waltham, MA 02454-9110. E-mail: jeff{at}brandeis.edu.

1 The abbreviations used are: RNAP, RNA polymerase; TEC, transcription elongation complex; BCCP, biotin carboxyl carrier protein; TPM, tethered particle motion; BSA, bovine serum albumin. Back

2 O. K. Wong, M. Guthold, D. Erie, and J. Gelles, manuscript in preparation. Back

3 C. Chan and R. Landick, unpublished data. Back

4 T. T. Perkins, H.-W. Li, R. V. Dalal, J. Gelles, and S. M. Block, submitted for publication. Back


   ACKNOWLEDGMENTS
 
We thank Jean Pyun and Oi Kwan Wong for instruction and advice.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
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Copyright © 2004 by the American Society for Biochemistry and Molecular Biology.
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