Optical tweezers studies of transcription by eukaryotic RNA polymerases

Introduction

Transcription of the genetic information from DNA into RNA is the first step of gene expression and a central point of cellular regulation. Transcription is performed by macromolecular enzymes called RNA polymerases (RNAPs) that move stepwise along the DNA template and produce a complementary RNA molecule. The process is traditionally divided into three main phases: initiation, in which the polymerase binds to a specific sequence in the DNA and starts the synthesis of RNA; elongation, during which the polymerase moves on the DNA template and produces a complementary RNA sequence; and termination, in which the polymerase dissociates from the DNA and releases an RNA transcript. All phases of transcription are highly-regulated processes with specific transcription factors acting together with the enzyme to make transcription a highly efficient process.

Gene transcription has been investigated with a number of different techniques. Classic biochemical assays gave insight into elongation dynamics of RNAPs and have identified proteins involved in transcription; structural studies gave detailed, albeit static, intermediate structures of RNAPs and accompanying transcription factors [reviewed in Ref. (1)]; and high-resolution genomic approaches and imaging techniques provided the kinetics of the polymerase movement and the dynamics of co-transcriptional processes, such as splicing and chromatin remodelling [reviewed in Refs. (2), (3)].

With the development of single-molecule biophysics techniques, it has become possible to observe and manipulate individual polymerases with very high spatial and temporal resolution. There are several advantages of using single-molecule techniques to study transcription dynamics. First, these techniques provide the ability to simplify the complex in vivo transcription machinery and to track and manipulate individual RNA polymerase molecules, one at a time, without the myriad of additional interactions that take place inside a cell (4), (5). Second, they enable access to the dynamics of single molecules, and thus allow us to circumvent the necessity to average over thousands of molecules in bulk experiments (4), (5), (6). Third, with single-molecule techniques it is possible to measure highly dynamic and fast movements, given the high spatio-temporal resolution that has become available (7). Fourth, individual molecules can be perturbed and manipulated in specific ways by force, and the response of an individual molecule to applied external force can be recorded (8), (9), (10), (11), (12). Therefore, single-molecule techniques enable the acquisition of precise and quantitative data, which allows the verification of biophysical theoretical models of transcription dynamics and regulation. This is needed for unravelling the complete spectrum of micromechanical events that underlie this fundamental biological process.

In this review, we focus on single-molecule optical tweezers studies of transcription [for a detailed review of other single-molecule techniques in transcription studies, please refer to Ref. (5)]. We give a brief summary of the methodology, give an overview of the past studies of transcription by bacterial and eukaryotic RNA polymerases and focus on recent advances in the field. The review is structured according to the phases of the transcription process: initiation, elongation and termination. We conclude by presenting current challenges of the optical tweezers technique, discuss future directions and consider potential integration with other techniques.

Basics of optical tweezers

Recent advances in physics and biology have led to the development of different single-molecule micromanipulation and detection techniques, such as single-molecule FRET measurements, optical and magnetic tweezers, atomic force microscopy, single-molecule fluorescent RNA detection methods. In this review, we focus on optical tweezers.

Optical tweezers are an instrument that uses highly-focused laser light to trap micron-sized particles. The basic principle behind optical trapping is momentum conservation, and this can be illustrated best in the ray-optics regime. Light is composed of photons that are retarded in media that are optically more dense. If one takes a small dielectric particle with an increased index of refraction as compared to the surrounding medium, photons are refracted as they enter and exit the particle due to the retardation of photons in the optically denser medium. This decrease in light velocity causes a change in the direction at the interface, and thus a change of the momentum of photons. Due to momentum conservation, however, the dielectric particle must experience an equal and opposite change of momentum. In a highly focused laser beam of sufficient power, this can give rise to an attractive tracking force that will capture the dielectric particle in three-dimensional (3D) space. More precisely, gradient forces act to restore the position of the particle towards the centre of the focused laser beam. The photons are not only refracted but also reflected from the surface of the particle, giving rise to scattering forces that push the particle in the direction of propagation of light (13), (14). It is due to scattering forces that the dielectric particle is trapped slightly downstream of the centre of the beam.

To achieve stable trapping in three dimensions, a single laser beam is typically focused through a high numerical aperture lens. For small distances around the centre, the trap behaves like a linear Hooke’s spring. The generated forces are proportional to the displacement of the object, according to F=kx, where x denotes the displacement of the particle from the trap centre, F is the optical force, and k is an effective stiffness of the trap. Knowing the trap stiffness is, therefore, crucial for accurately determining the forces involved and is usually determined from thermal fluctuations of the position of the trapped particle [i.e. Brownian motion calibration; Refs. (15), (16), (17)]. Forces typically exerted by optical tweezers are on the piconewton level (pN) and distances measured are on the nanometre level (nm). These length- and force-scales make optical tweezers particularly useful for investigating the functions of cytoplasmic and DNA molecular motors, such as RNA polymerases.

In biological applications, the forces are usually not applied directly on the macromolecules of interest, but on glass or polystyrene micron-sized beads that are linked to them. Different experimental geometries were used in optical tweezers transcription experiments, e.g. single optical traps with macromolecules tethered directly to the surface of the chamber (18), (19) or systems where one particle is trapped in an optical trap and another one is held with a micropipette (20). Finally, the dual-trap experimental system uses a single trapping laser that is split by polarisation into two beams, each of which forms an optical trap. This configuration reduces the noise in the system, as beads are not attached to any surface, but are trapped free in solution. The data is recorded in each trap independently, thus only the anti-correlated data represents the signal and the correlated data can be attributed to noise (6).

In a typical dual-trap optical tweezers transcription experiment, the DNA tether is formed between the tagged polymerase attached to one bead (usually via a biotin-streptavidin interaction) and the tagged end of the DNA attached to another bead (usually via a digoxigenin-anti-digoxigenin interaction) (Figure 1A). Depending on where the tagged DNA is located with respect to the polymerase, i.e. upstream or downstream of the polymerase, the forces applied on the enzyme can either assist or oppose the transcription. In the assisting force mode, the tagged DNA is upstream of the polymerase, therefore an active transcription by the enzyme increases the distance between the beads and reduces the force. In the opposing force mode, the DNA is labelled downstream of the transcribing polymerase, which results in decrease of the distance between the beads and increase of the force. Both configurations are used in optical tweezers transcription experiments. Opposing force mode is preferred for studying pausing and backtracking dynamics of RNA polymerases, as it was shown that the enzymes pause more with opposing forces (11), (21). Assisting force mode is often used in studies of transcription through nucleosomes, where the tension applied to the upstream DNA ensures there is no transfer of nucleosomes behind the polymerase (22), (23), (24).

Figure 1:

Dual-trap optical tweezers experimental set-up scheme and a representative RNA polymerase trajectory.

(A) Two optical traps (grey) are used to trap functionalised polystyrene beads, typically around 2 μm in diameter (green). Tagged polymerase is attached to one bead and the labelled DNA is attached to another bead. In the opposing force mode experimental set-up shown here, the polymerase transcribes the DNA in between the two beads, therefore shortening the bead-to-bead distance during the experiment, while the forces applied to the enzyme are increased. Note: the scheme is not to scale. (B) Example optical tweezers transcription trace showing transcribed nucleotides over time of an individual transcribing RNA polymerase I. The raw unfiltered data is shown in black, with the filtered trace overlaid in white. Pauses are marked in red.

Studies of transcription initiation

Transcription initiation is a highly regulated process during which an RNA polymerase binds to a promotor sequence of a gene and unwinds double-stranded DNA around the transcriptional start site to form a transcription bubble. Initiation in prokaryotes is assisted by a single transcription factor – σ factor, while in eukaryotes it requires a myriad of proteins (25). Due to the complexity of the pre-initiation complex (PIC) in eukaryotes, until recently, most of the single-molecule studies focused on prokaryotic transcription initiation. Studies using methods such as single-molecule FRET and magnetic tweezers have showed that abortive initiation of bacterial RNA polymerase proceeds mainly through a scrunching mechanism, in which the downstream DNA is unwound and pulled into a stationary RNAP (26), (27) and that most mature elongation complexes retain σ70 factor throughout elongation (28). Furthermore, recent work revealed dynamics of the RNAP initiation complex (29) and the pausing of RNAP during initial transcription at lac promoters (30).

A recent study by Fazal and colleagues reported an extraordinary achievement of assembling a 32-protein pre-initiation complex (PIC), in real time, using optical tweezers to study the initiation of yeast RNA polymerase II (Pol II) (10). The complex consisted of Pol II and six general transcription factors, including TATA-binding protein, TFIIB, TFIIE, TFIIF, TFIIH and TFIIA and Sub1 (Figure 2A). The authors found that during initiation a large bubble is opened in the DNA, driven by a member of the PIC, the TFIIH helicase. Contrary to previous findings that Pol II produces only a short transcript during initiation, Fazal and colleagues found that Pol II synthesises 85 bp on average, up to the length of the open bubble, followed by promotor escape (Figure 2B). These studies from yeast are likely to hold true even in metazoans, as it was shown in several species that the transcriptional start sites are located far from the TATA-boxes, therefore requiring a bigger bubble.

Figure 2:

Transcription initiation by Pol II in assisting force mode.

(A) Schematic diagram of the transcription initiation assisting force mode set-up, where a tether is formed between the two beads (blue), with Pol II attached to one bead, and the upstream DNA attached to the second bead. As transcription proceeds, the distance between the beads increases. All proteins used for initiation are depicted here, except Sub1. The scheme is not to scale. (B) Representative traces of Pol II elongation, where red arrows mark the promotor escape and the black arrows mark the increase of force that was used to confirm elongation. Adapted from Ref. (10) with permission from Nature Publishing Group.

Elongation phase

After successful initiation, the RNA polymerase, the DNA and the nascent RNA form a stable transcription elongation complex (TEC) that moves downstream on the DNA, in the 5′ to 3′ direction, while incorporating complementary nucleotides in the growing RNA chain. A number of single-molecule studies provided great insights into the dynamics of transcription elongation, pausing, elongation in the presence of transcription factors and through nucleosomal templates.

Transcription through nucleosomes

The genetic material of eukaryotic cells is organised into chromatin, a complex of DNA wrapped around histone proteins forming nucleosomes. It was proposed by bulk experiments that nucleosomes represent an energetic barrier to elongating Pol II (75), (76), (77). Several optical tweezers studies further investigated how Pol II overcomes nucleosomes during elongation and successfully transcribes eukaryotic genes.

Hodges and colleagues used optical tweezers to study transcription of Pol II through a single nucleosome template (Figure 3A) (22). Their studies confirmed that a nucleosome behaves like a barrier that both decreases the rate of forward translocation of Pol II and increases pausing and backtracking. Furthermore, they found that the progression of the enzyme through the nucleosome depends on nucleosomal fluctuations, i.e. nucleosomal breathing or local unwrapping of the nucleosomal DNA due to thermal fluctuations. The nucleosome unwraps the DNA locally, thereby giving the polymerase enough DNA to transcribe through. Bintu and colleagues further investigated the influence of specific histone modifications and nascent RNA secondary structure on transcription through nucleosomes (23). They showed that the local nucleosome unwrapping dynamics, and in turn the transcription elongation dynamics, is affected by nucleosomal properties such as the histones modification state, presence of histone tails or the underlying DNA sequence.

Figure 3:

Transcription through nucleosomal DNA.

(A) Transcription traces of individual Pol II transcribing through a single nucleosome template with different ionic strengths. The presence of a nucleosome represents a barrier to transcription, causing long pauses and arrest of Pol II. (B) Transcription traces of individual Pol II transcribing through dinucleosomal DNA templates, at 50 bp and 45 bp distance. The presence of a second nucleosome at a 50 bp distance from the first one impairs Pol II transcription dynamics through the first nucleosome. (C) (Upper) Passage probabilities of Pol II through the first nucleosomal region: Pol II transcribes more efficiently through two nucleosomes at 45-bp than at 50-bp distance. (Lower) Angular distributions of the two neighbouring nucleosomes for the 50-bp (nucleosomes face the same side of the DNA) and 45-bp (nucleosomes face opposite sides of the DNA) linker templates. Adapted from Refs. (22), (24) with permission from AAAS and USA National Academy of Sciences.

Recently, our group used optical tweezers to study transcription of Pol II through nucleosomal templates of higher complexity and the influence of nucleosomal geometry on the efficiency of transcription (Figure 3B) (24). Fitz and colleagues investigated Pol II elongation dynamics along a DNA template with two nucleosomes. Our work showed that the presence of a second nucleosome affects the probability of the polymerase to pass through the first nucleosome. This effect is dependent on the spacing between the two nucleosomes and their rotational arrangement along the DNA (Figure 3C). Pol II could transcribe more efficiently when the second nucleosome was facing opposite side of the DNA template, suggesting that the 3D nucleosomal arrangement affects Pol II transcription dynamics.

Elongation with transcription factors

The dynamics of transcription elongation is regulated by different transcription factors, such as TFIIS and TFIIF in the case of eukaryotic Pol II. The elongation factors act through various mechanisms, increasing overall transcription rates or by affecting proofreading and fidelity and by reactivating backtracked polymerases. Optical tweezers assays are particularly suitable for studying the influence of individual transcription factors on polymerase elongation, as they allow timely-controlled addition of purified factors to the transcribing elongation complex.

One of the most studied transcription factor in optical tweezers assays is TFIIS. This protein is known to enhance the cleavage of backtracked RNA and recovery of backtracked Pol II (9), (11), (35), (50), (52), (53), (56), (64). Without TFIIS, Pol II can only transcribe up to forces of 7.5 pN, before entering a backtrack. However, by rescuing backtracked polymerases, TFIIS enables Pol II to transcribe up to 16.9 pN (Figure 4A) (56). Therefore, TFIIS enables Pol II to transcribe against higher loads, increasing its mechanical performance, which in turn allows it to transcribe through nucleosomes more efficiently (35). TFIIS acts mainly by affecting the pausing dynamics of Pol II, decreasing the time the enzyme spends in a pause and not by affecting its overall transcription rate (35). Two different studies investigated the dynamics of TFIIS-assisted reactivation of backtracked Pol II (9), (11). By using a cleavage-defective mutant, Schweikhard and colleagues showed that at low force TFIIS can relieve arrested Pol II in a cleavage-independent way. The authors argued that this might be in accordance with structural results that show that TFIIS weakens the interactions of backtracked RNA and Pol II, by competing for the same binding sites inside the Pol II funnel (52). Furthermore, the work from our group showed that TFIIS increases the backtrack recovery efficiency of Pol II. TFIIS enables rapid recovery from backtracks of any depth and even when it associates with an already backtracked enzyme (11).

Figure 4:

Elongation in the presence of transcription factors.

(A) Transcription traces of Pol II in the presence and absence of TFIIS. TFIIS rescues backtracked Pol II and enables it to transcribe to higher forces. (B) Arrest probabilities of Pol II in a nucleosome region (marked in yellow), in the presence of TFIIS, TFIIF or both. Both TFIIS and TFIIF enable Pol II to transcribe more efficiently through nucleosomes, as seen with the increased percentage of passages through the nucleosome region. Adapted from Refs. (11), (35) with permission from USA National Academy of Sciences.

Another transcription factor affecting the dynamics of Pol II elongation is TFIIF. Apart from being a general transcription initiation factor, TFIIF was shown to enhance overall transcription rates of Pol II (78), (79). Optical tweezers experiments confirmed that TFIIF enhances elongation efficiency of Pol II (Figure 4B), but not through changing pause-free velocity of the enzyme, but by decreasing the frequency and the duration of Pol II pausing during assisting force mode assays (35). Furthermore, Schweikhard and colleagues found that TFIIF stimulates the restart of the backtracked Pol II in a low-force regime. However, at high forces there is no effect of TFIIF alone on Pol II backtrack recovery, although it enhances the effects of TFIIS. The authors argued that TFIIF could interact with TFIIS and in this way stabilise it on the backtracked Pol II complex.

Termination

Termination is the final stage of transcription during which a transcribing polymerase and the nascent RNA are released from the DNA. Termination defines boundaries of transcription units, ensuring there is no interfering with the transcription of the downstream gene and securing there is enough RNA polymerase ready for reinitiation. Termination in prokaryotes can be mediated through terminator sequences within the nascent RNA that form a stem-loop structure and cause the RNA polymerase to stop and dissociate; or through a Rho-dependent mechanism, in which a Rho protein binds to the RNA and runs towards the RNAP (80). Termination mechanisms in eukaryotes are more complex and remain poorly understood (81), (82), (83), (84). Therefore, optical tweezers studies thus far focused only on prokaryotic termination.

In both mechanisms of prokaryotic transcription termination, the release of the polymerase is caused by the forces exerted on the RNA, either by folding of the RNA hairpin or by Rho displacement, which results in forward translocation of the polymerase without the RNA synthesis. Therefore, optical tweezers studies focused on applying external forces on the nascent RNA. Dalal and colleagues performed experiments with an experimental geometry that allows pulling on the elongating RNA chain (8). They found that RNAP could transcribe up to forces of 30 pN applied to the RNA, which demonstrates high TEC stability and implies that loads required to terminate elongation have to be higher than this level. Larson and colleagues used similar RNA and DNA pulling assay to investigate the transcription efficiency of different terminator sequences that differ by the size of the hairpin and the length of the U-tract (85). They found that termination occurs through a combination of shearing and forward translocation mechanisms, depending on the composition of the U-track of the terminator sequence.

A recent study by Koslover and colleagues used optical tweezers to investigate the interaction of the Rho protein with the nascent RNA (86). The authors used a dumbell assay that consisted of an RNAP that was attached to the surface of a streptavidin bead and the nascent RNA that was annealed to a DNA handle, which was attached to another bead. In this configuration, the Rho protein binds to the nascent RNA and in the presence of ATP translocates towards the RNAP. Detachment of Rho from the RNA was seen as a rip in the force-extension curves. They found that Rho can adopt two RNA binding states, one that binds around 57 nt and one that binds 85 nt. Furthermore, they suggested that Rho translocates towards the RNAP by a tethered-tracking mechanism, looping out the RNA between Rho and the RNAP. The assay is suitable for further studies of transcription termination, studying the step size of Rho protein and the interaction with its cofactors, NusA and NusG.

Conclusions and outlook

Optical tweezers studies have in the last two decades revealed numerous micromechanical details of the transcription process. They have proven to be an invaluable tool to study gene transcription. Initially, optical tweezers studies investigated viral or bacterial enzymes running on the DNA template. However, over the years, they have become increasingly more complex, probing the functions of different eukaryotic RNA polymerases, numerous transcription factors and considering complex 3D DNA/nucleosome arrangements. A key feature of all these studies lies in the fact that optical tweezers measure actual trajectories of individual enzymes, providing a wealth of details on the dynamics of the process. The quantitative nature of optical tweezers data makes it an ideal tool for testing various theoretical models of translocation, pausing, and recently as an example of a stochastic resetting process (72). On the side of physics, the implications of utilizing a template for polymerizing a copolymer (RNA) on the dynamics of the process are just beginning to become grasped (87). Furthermore, as theoretical models become more and more refined, the amount of single molecule data required for comparing and distinguishing between them increases. New developments are needed on the experimental side in order to achieve this, perhaps by switching to fully automated optical tweezer experiments.

However, many questions remain open. The largest eukaryotic RNA polymerase, RNA polymerase III, has not been studied with optical tweezers. Comparing transcription dynamics of the three eukaryotic polymerases will provide insights into the evolution of these enzymes. In particular, the cleavage activity of the three eukaryotic polymerases was shown to be differently regulated (66). Obtaining data on backtracking dynamics and recovery of Pol III would provide further insights into the nature of transcriptional pausing in eukaryotes. Much of the transcription studies thus far have been performed on plasmid DNA. It would be interesting to use actual gene templates in the future, to investigate the influence of the underlying sequence and gene features on transcription dynamics.

Finally, future studies of transcription will greatly benefit from combining optical tweezers with other techniques. In combination with fluorescence microscopy, optical tweezers were used in DNA overstretching experiments and for determining DNA-protein interactions (88), (89), (90). Angular optical trapping (AOT) was developed for simultaneous detection of force and torque (91), (92), which enabled studying transcription dynamics and nucleosome stability under conditions of applied torque on the DNA (93). Recent advancements in combining optical tweezers with confocal microscopy, as well as development of advanced microfluidics systems will likely increase the range of applications and enable investigation of conditions more similar to the in vivo situation.

To conclude, much has been learned, but we are still far from a detailed understanding of how the information that is contained within DNA is translated to RNA form. Optical tweezers studies will contribute to the future advances in the field.