Promoter melting triggered by bacterial RNA polymerase occurs in three steps

Abstract

RNA synthesis, carried out by DNA-dependent RNA polymerase (RNAP) in a process called transcription, involves several stages. In bacteria, transcription initiation starts with promoter recognition and binding of RNAP holoenzyme, resulting in the formation of the closed (R·P_c) RNAP-promoter DNA complex. Subsequently, a transition to the open R·P_o complex occurs, characterized by separation of the promoter DNA strands in an approximately 12 base-pair region to form the transcription bubble. Using coarse-grained self-organized polymer models of Thermus aquatics RNAP holoenzyme and promoter DNA complexes, we performed Brownian dynamics simulations of the R·P_c → R·P_o transition. In the fast trajectories, unwinding of the promoter DNA begins by local melting around the -10 element, which is followed by sequential unzipping of DNA till the +2 site. The R·P_c → R·P_o transition occurs in three steps. In step I, dsDNA melts and the nontemplate strand makes stable interactions with RNAP. In step II, DNA scrunches into RNA polymerase and the downstream base pairs sequentially open to form the transcription bubble, which results in strain build up. Subsequently, downstream dsDNA bending relieves the strain as R·P_o forms. Entry of the dsDNA into the active-site channel of RNAP requires widening of the channel, which occurs by a swing mechanism involving transient movements of a subdomain of the β subunit caused by steric repulsion with the DNA template strand. If premature local melting away from the -10 element occurs first then the transcription bubble formation is slow involving reformation of the opened base pairs and subsequent sequential unzipping as in the fast trajectories.

The DNA-dependent RNA polymerase (RNAP), whose sequence, structure, and functions are universally conserved from bacteria to man (1, 2), is the key enzyme in the transcription of the genetic information in all organisms (3–5). There are three major stages in the transcription cycle (3), which first requires binding of promoter-specific transcription factors to the catalytically-competent core of RNAP, to form a holoenzyme. They are: (i) initiation, which first requires binding of an initiation-specific σ factor to the catalytically-competent core RNAP to form the holoenzyme, followed by recognition of the promoter DNA to form the closed (R·P_c) complex and subsequent spontaneous transition to the open (R·P_o) complex; (ii) elongation of the transcript by nucleotide addition; (iii) termination involving cessation of transcription and disassembly of the RNAP elongation complex. Among these highly regulated stages, the most complicated may be the initiation process because it involves promoter recognition, DNA unwinding, and the formation of the transcription bubble inside the RNAP active-site channel, where RNA synthesis occurs.

A simplified transcription initiation pathway is (6, 7)

[1]

where R is RNAP, P is the promoter DNA (Fig. 1A), and R·P_c and R·P_o are the closed and open complexes (Fig. 1B), respectively. IC_≤12 is the abortive initiation complex with transcript size ≤ 12 nt, and TEC is the transcription elongation complex. Here, we focus only on the dynamics of transcription bubble formation, which occurs during the R·P_c → R·P_o transition. The approximately 150 Å long and 110 Å wide core enzyme from the bacterial Thermus aquaticus (8, 9) has five subunits, αI, αII, β, β′, and ω (Fig. 1B) that are assembled like a crab claw. The two “pincers,” formed from the large β and β′ subunits, hold the promoter DNA in the active-site channel between the pincers (Fig. 1). The structural model of RNAP holoenzyme complexes with fork-junction DNA (10) has given insights into the mechanism of the R·P_c → R·P_o transition. A variety of structures were pieced together to construct detailed models for the R·P_c and R·P_o, which lead to the following mechanism of R·P_o formation (3, 10). Local melting of the promoter -10 element allows binding of the exposed nontemplate strand to conserved region 2.3 of σ (σ_2.3) (Fig. 1B), stabilizing the melted region. Melting also renders the promoter DNA flexible, thus facilitating its entry into the active channel. The R·P_o structure further suggests that the promoter DNA bends into RNAP active channel to form the transcription bubble (11–13). Although the structural models provide plausible hypothesis for the transcription bubble formation, dynamical studies are required to describe the conformational changes that accompany the R·P_c → R·P_o transition.

Fig. 1.

Structural models for the promoter DNA and RNAP. (A) Schematics of the base pairing between the template (green) and nontemplate (yellow) strands of the promoter. Nucleotide positions are numbered relative to the transcription start site, +1. DNA segments that interact with RNAP, -35 and -10 elements, are shaded red. (B) Structural models on top correspond to R·P_c (left) and R·P_o (right) and are color-coded as β cyan, β′-magenta, σ-orange, αI, αII, and ω-gray, DNA nontemplate strand-green, and template strand-yellow. The β subunit is removed to show the interactions of the promoter with the σ subunits (bottom left) and the transcription bubble structure on the bottom right, while on the top row it is shown in full opacity.

Here, we use the structural models for R·P_c and R·P_o (updated from (10)) of T. aquaticus to address the following questions: (a) What are the steps in transcription bubble formation, and (b) What is the nature of RNAP dynamics that enables the downstream dsDNA entry into the enzyme? To answer these questions, we performed Brownian dynamics simulations of the R·P_c → R·P_o transition using a coarse-grained SOP model (14). The simulations of the kinetics of the R·P_c → R·P_o transition show that the transcription bubble forms in three distinct steps. In step I, a region near the -10 element on the promoter DNA melts. In step II, the promoter DNA scrunches into the RNAP active channel, forming the transcription bubble and in step III, the downstream DNA bends. DNA bending is a result of downstream DNA relaxation, and occurs only after unwinding of the dsDNA. Widening of the channel needed to accommodate the dsDNA entry into the active channel requires transient expansion of key structural elements in the β subunit of RNAP, which implies that the internal enzyme dynamics plays an important role in the R·P_c → R·P_o transition.

Results

A Network of Contacts Trigger the Promoter Melting.

The structures of the RNAP-DNA complexes used in this work (10) have 3,122 residues and 150 nucleotides. The holoenzyme has six subunits: αI (Ala6-Glu229), αII (Ala6-Phe225), β (Ala2-Ala1116), β′ (Ala3-Ala1499), ω (Ala2-Ala93), and σ (Ala93-Ala438) (Fig. 1B). The promoter DNA has 75 base pairs, -54 to +21, labeled with respect to the transcription start site +1 (Fig. 1A). In R·P_c, the promoter DNA “sits” on the top of RNAP (Fig. 1B) and forms stable interactions between the σ subunit and the -10 and -35 regions. Despite the low-resolution nature of the models, the dynamics reveal key structural changes that occur during the R·P_c → R·P_o transition. Several contacts between DNA -10 element and the subunit σ of RNAP rupture, in particular, the contacts involving nucleotides -12 to -8 on the template strand (Fig. S1). Formation of contacts between nucleotides -3 to +5 on the nontemplate strand and the β subunit (Fig. S1), nucleotides -9 to +1 on the template strand and the β subunit (Fig. S1), and nucleotides +1 to 7 on the template strand and the β′ subunit (Fig. S1) stabilize the R·P_o state.

R·P_c → R·P_o Transition Trajectories Partition into Fast (Efficient) and Slow (Inefficient) Tracks.

The global nature of the R·P_c → R·P_o transition is monitored by the time-dependent changes of the root mean square deviation Δ_C(t) (Δ_O(t)) of the DNA with respect to the closed (open) value. The transition times, τ_ms, calculated using |Δ_C(τ_m) - Δ_O(τ_m)| < ϵ = 0.1 Å, were used to partition the set of 30 trajectories into fast and slow processes (Fig. S2). In the fast routes, Δ_C(t) (Δ_O(t)) increases (decreases) very rapidly (Fig. S2), which indicates (see below) that upon opening of the DNA base pairs the transcription bubble forms efficiently. In contrast, in the slow routes, long-lived metastable states, which are indicated by a plateau in Δ_C(t) (Δ_O(t)) (Fig. S2), are populated. The base pairs open by a complicated pathway resulting in a decreased efficiency in the transcription bubble formation. The conclusions do not depend on the value of the dielectric constant used in the treatment of the electrostatic interactions (see Eq. S1 in Figs. S2 and S3).

What is the origin of the differences between the fast and slow trajectories? To answer this question, we examined the time-dependent conformational changes of the promoter DNA, which we describe using the distances, d_i(t), between the two complementary nucleotides of each base pair in the promoter DNA. Here, where and are the positions of the ith nucleotide on the template and the nontemplate strands, respectively. Since the transcription initiation site is at i = +1 and the transcription bubble forms between i = -12 and +2 (6), we computed d_i(t) for four representative base pairs, -11, -7, -3, and +2 to describe the transcription bubble formation. In the R·P_c state, d_i(t) ≈ 11 Å for all of the four base pairs, but in R·P_o state, d_i(t) ≈ 18 Å, 48 Å, 53 Å, and 28 Å for -11, -7, -3, and +2 base pairs, respectively.

Promoter DNA Unzips Sequentially from -10 Element in the Fast Track.

Analysis of d_i(t) in all the fast trajectories shows a consensus sequence of events during the R·P_c → R·P_o transition. Base-pair -11 opens first at t ∼ 10 μs, which is followed by disruption of interactions in -7 at t ∼ 16 μs. In both cases the equilibrium values corresponding to the structure in R·P_o were reached rapidly (Fig. 2, Fig. S4). At t ∼ 24 μs, the -3 pair rips and the distance between the nucleotides attains the value in the R·P_o state. The distance d₊₂(t) fluctuates between 11 Å and 30 Å, and reaches 30 Å shortly after base-pair -3 opens. The opening of the upstream base pairs favor the rupture of the downstream neighbors, which establishes that the base pairs from -12 to +2 rupture abruptly in a sequential manner by an unzipping mechanism. Complete analysis of all the d_i(t)s shows that sequential unzipping starting from the -10 site leads to rapid transcription bubble formation.

Fig. 2.

Dynamics of transcription bubble formation. (A) Distance between complementary nucleotides of the base pairs in the bubble region as functions of time for a representative fast trajectory. Cyan, red, black and blue lines correspond to d_i(t) for base pairs -11, -7, -3, and +2, respectively. Structures of the RNAP complex at t = 12 μs, 16 μs, and 24 μ during the development of the transition bubble are shown. (B) Same as (A) except the results are for a slow trajectory. The reformation of the prematurely opened base pairs -3 and 2 (black and blue curves respectively) is highlighted in the shaded region. The structures sampled during the R·P_c → R·P_o transition are highlighted. Arrows indicate the starting and ending states.

Initial Base Pair Opening Away from the -10 Element Results in Slow R·P_c → R·P_o Transition.

Although promoter recognition sequences are localized in the -35 and -10 regions, melting of the base pair can occur stochastically (see also Fig. S5). However, as seen in the fast trajectories for efficient transcription bubble formation, rupture must start from the -10 element. In some trajectories, multiple base pairs melt nearly simultaneously in a nonsequential process, which greatly impedes transcription bubble formation. Fig. 2 shows that in one of the slow trajectories, rupture of base-pairs -3 and +2 compete with the opening of the base-pair -7. At t ∼ 40 μs, base-pair -7 remains intact while the downstream base-pairs -3 and +2 rip as seen in the increase of d_-3(40 μs) and d₊₂(40 μs) from 11 Å to 40 Å and 11 Å to 50 Å, respectively (Fig. 2). At longer times, the base-pairs -3 and +2 reform as shown by the decrease in d_-3 and d₊₂ to 11 Å, the value in the R·P_c state, resulting in the “resetting” of the initial state. Subsequently, melting of the base pairs occur sequentially, which is manifested in the increase of d_i(t) to the values in the R·P_o state.

Transcription Bubble Formation in Fast Trajectories Occurs in Three Steps.

We used the time-dependent distance changes between the ith (jth) nucleotide on the promoter sequence and jth residue on RNAP, , () is the position of the ith nucleotide (residue), to identify three steps in the transcription bubble formation process. The overall dynamics associated with the bubble formation is assessed using d_-5(t), which on an average (black line in Fig. 3A) occurs in about (30–40) μs. Dissection of the events leading to the increase in d_-1(t) from about 11 Å at t = 0 to 55 Å at t ≈ 35 μs shows that transcription bubble formation occurs in three major steps.

Fig. 3.

Three steps (in shaded colors) in the transcription bubble formation in the fast trajectories. (A) The time-dependent increase in d_-5(t) shows the growth of the transcription bubble. The black line shows d_-5(t) averaged over multiple trajectories, and the individual traces for a few trajectories are shown for comparison. (B) Average changes in the distance between the nucleotide -12 on the nontemplate strand and Mg²⁺ as a function of time. (C) Ensemble average changes in d_ij(t) for i = -12 and j = +21 as a function of t. (D) The time-dependent change in the angle Ψ between the interaction centers at -35, -10, and +21 shows that only after transcription bubble forms (see (A), the dsDNA bend (Ψ decreases in step III, which is shaded in light blue color). (E) Schematic representation of dynamical processes in (A)–(D). The sketches from top to bottom correspond to bubble growth (d_-5(t) increase), promoter melting (decrease in d_-5,Mg²+(t)), DNA scrunching (decrease in d_-12,+21(t)), and dsDNA bending (decrease in Ψ(t)). On top right are shown the front view of R·P_c (left) and R·P_o (right) structures without the β subunit and a few relevant nucleotides are labeled for reference.

Step I: The -10 element melts locally.

The decreases in d_{-5^′,Mg²⁺}(t) (the prime refers to the base-pair number on the NT strand) (Fig. 3B) shows that the nontemplate strand moves towards the active site of the enzyme (amino acids around the Mg²⁺ ion). In this stage, d_-12,+21 also decreases (Fig. 3B), but in contrast to d_{-5^′,Mg²⁺}(t), the changes in the conformations of +21 base pair relative to the RNAP continues to evolve throughout the transcription bubble formation process (see below). Compared to the time needed for the bubble opening (Fig. 3A), the characteristic time associated with the decrease of d_{-5^′,Mg²⁺}(t) (t ∼ 16 μs) is short, and is the major feature of step I. Although the template strand of the promoter DNA also moves synchronously in this step the dynamics of such a process lasts over the entire duration of bubble formation (at t ∼ 35 μs). Therefore, we do not consider the movement of the template strand as a major feature of step I. The correlated movement of the nontemplate and the template strands is consistent with the assumption that local melting of DNA in the -10 element renders it flexible. In addition, the observed stabilization of the nontemplate strand in step I agrees with the experimental finding that the conserved aromatic residues of σ are positioned to recognize and stabilize the exposed nontemplate strand (15–17). Loss of base-pair interaction upon melting of the -10 base pairs (TATAAT) results in the formation of favorable interaction of adenine or thymine nucleotides with the aromatic side chains (Phe248, Tyr253, and Trp256) on σ2.3 subunit as well as electrostatic interactions between DNA and the enzyme.

Step II: DNA scrunches into the RNAP active-site channel and forms a bubble.

The transcription bubble starts to grow from the -10 to +2 base pair as the promoter sequence unzips. The bubble region quantified in terms of center, d_-1(t), increases from 11 Å to 55 Å, the value in the R·P_o state, which implies that in this step promoter DNA unwinds and the strands separate. Meanwhile, the distance between the -10 element and the downstream edge of the promoter DNA (base-pair +21) decreases from 110 Å to 55 Å (see the V-shape d_-12,+21 in Fig. 3C). The observed decrease in Fig. 3C during step II is reminiscent of the scrunching mechanism proposed for the R·P_o → R·P_itc based on single molecule FRET experiments (10, 18). According to the scrunching mechanism, in each cycle of the abortive initiation, RNAP pulls the downstream DNA into the active channel and past its active center without substantial change in the enzyme conformation (18, 19). Although the R·P_itc transition formation occurs only after the completion of the R·P_c → R·P_o transition, the scrunching mechanism appears to also drive the formation of the transcription bubble (see below).

Step III: Downstream DNA bends.

In the final stage, the downstream DNA bends to release the strain accumulated during the promoter DNA opening. The strain release results in the increase in d_-12,+21(t) after the decrease in step II (Fig. 3C). In addition, DNA bends and kinks in the downstream region, which is captured by the time-dependent changes in the angle, Ψ(t) (Fig. 3D). The angle Ψ(t) formed between the interacting sites localized at -35, -12, and +21, which is relatively constant during step I and II, decreases in step III from ∼150° to ∼100°, the value in the RP_o state (11–13).

Our finding that DNA bending occurs only after the promoter DNA opening and bubble formation is in apparent conflict with the structural model that DNA bending facilitates entry into the active channel of the enzyme (3). Indeed, it is not realistic for DNA to bend or kink across the entrance of the active-site channel for the following reasons: (i) We expect that the flexibility in DNA, which is caused by local melting around -10 region, allows bending to be nearly isotropic restricted only by volume excluded by RNAP; therefore, it is entropically unlikely for DNA to bend in a specific direction and enter the active channel. (ii) Because DNA is negatively charged it should be repelled by the negatively charged σ 1.1 (20, 21). The location of σ 1.1 makes it unlikely for DNA to bend into the RNAP active channel prior to unzipping. Since the relaxation of the stressed DNA occurs after the opening of the promoter DNA, the observed DNA bending in the R·P_o state must be associated with the third stage of the bubble formation process.

Scrunching is a universal mechanism in transcription initiation.

To illustrate whether the promoter DNA scrunches into RNAP in the R·P_c → R·P_o transition, we calculated the time-dependent distance changes, d_ij(t), for the same sites that are labeled in the FRET study (18, 22) (Fig. 4). The conclusion that during the R·P_c → R·P_o, the promoter DNA is scrunched follows the arguments used in the experimental study (18). From the decrease in the distance between the leading edge of RNAP (residue 366 of σ) and the +20 site in downstream DNA (Fig. 4A), it follows that RNAP translocates relative to downstream DNA. The lack of distance changes d_σ569,-39(t) and d_σ569,-20(t) between the upstream edge of RNAP (σ569) and upstream sites in DNA (-39 and -20) during the course of R·P_c → R·P_o transition (Fig. 4 B and D) implies absence of translocation of RNAP relative to upstream promoter sites. Similarly, d_σ366,-20 is unchanged during the R·P_c to R·P_o transition, which implies that the leading edge of RNAP is stationary with respect to upstream DNA. Direct measurements of distance changes between site -15 and +15 on the promoter DNA shows that d_-15,+15(t) decreases by about 5 Å when the R·P_c → R·P_o transition is completed. These results show that, to a large extent, RNAP is fixed on the promoter while the distances between downstream edge of DNA decrease.

Fig. 4.

Dissection of DNA scrunching during Step II. Structures on the top right, top view of R·P_c (left) and R·P_o (right) structures are shown for reference. The distances between nucleotides of the promoter DNA and RNAP as function of time, d_ij(t) are on the left. In (A)–(D) the red dashed lines (solid green lines) are d_ij values in R·P_c (R·P_o) state. (A) Ensemble averaged distance between nucleotide +20 and the leading edge residue σ366 (numbering as in E. coli). (B) Change in the distance between the trailing head residue σ569 and nucleotide -39 as a function of t. (C) Time-dependent change in the distance between the leading edge residue σ366 and the upstream nucleotide -20. (D) Distance between the trailing edge residue σ569 and nucleotide -20 as a function of t. (E) Average changes in d_-15,+15(t) as a function of t. (F) The sketches from top to bottom capture the distance changes shown on the left. Structures on the top right, top view of R·P_c (left) and R·P_o (right) structures are shown for reference.

The “V” shape portion of d_σ366,+20(t) and d_-15,+15(t) (Fig. 4 A, E) shows that the distance changes associated with leading edge and the specific sites on DNA do not change monotonically in R·P_c → R·P_o transition. The distance changes between labeled sites on promoter DNA and changes in Ψ(t) (Fig. 3 C,E) show that the “V” shape of the distances is largely due to the contraction (or scrunching) of the DNA in the downstream region. The subsequent increase in the variables shown in Fig. 4 A, E is due to the DNA bending.

Active Channel of RNAP Opens by a “Rope-Swing” Mechanism.

Despite the enhanced flexibility of the melted region of DNA, it is unclear how the promoter gains access to the binding sites in RNAP. The main DNA binding channel in the RNAP holoenzyme (10–12 Å in diameter for Thermus thermophilus and 16–19 Å for Thermus aquaticus) is narrow compared to the 22 Å diameter of the dsDNA. (9, 23). Based on static structures alone, two models have been proposed to explain how the RNAP channel expands to accommodate the dsDNA. One possibility is that the repulsive interactions between the negatively charged σ1.1 domain and the dsDNA result in the opening of the channel (3). Alternatively, based on a high resolution structure of the holoenzyme in the closed state, it was suggested that the protruding region of the β-subunit could fit into dsDNA major groove (23). In particular, it was argued that the β-subunit loop serves as a “gate” that allows only single stranded DNA to go through, and the “gate” stabilizes the DNA orientation to favor melting (23). However, in the absence of the crystal structure of the intermediate states of transcription bubble formation or experimental evidence, the mechanism whereby the RNAP channel opens remains elusive. Our simulations of the transcription bubble formation process suggest that channel opening occurs by a “rope-swing” mechanism (see below) and is triggered by transient movements of structural elements of the β subunit.

Analysis of the structures reveals that the Ala132-Ser387 region of the β-subunit makes extensive contacts with the promoter DNA in R·P_o but not in R·P_c, which suggests that this region (referred to as the Rope-Swing (RS) region) must play an essential role in the channel widening process. To dissect the kinetic role of the RS region we partition it into the swing region (S), which is the N-terminal globular β2 domain (Ala132-Arg334) (24, 25) and the Rope (R) region, a part of the β1 domain corresponding to the C-terminal helix-turn-helix motif (Thr335-Ser387) (Fig. 5A). The R motif is the analogue of the helix-loop-helix fragment in T. thermophilus RNAP (23). The widening of the DNA binding channel is facilitated by the coordinated displacement of the R and S regions as shown by time-dependent changes in a number of quantities associated with the R and S regions as well as the β′ subunit. The closest distance, R(t), between the R and S regions, whose value is 14 Å in R·P_c, increases past the value in the final R·P_o state over a “gating” time interval Δτ_w that ranges from 19 μs to about 28 μs. Over the duration Δτ_w the changes in the distance d_AB(t) (A is the center of mass of the RS structural unit and B is the center of mass of the residues Ala105–Ala499 of the β′ subunit) as a function of t shows (Fig. 5C) a total increase of 7 Å during the R·P_c → R·P_o transition. The 7 Å increase, which occurs transiently (Fig. 5 B, C), is sufficient to enable entry of the dsDNA. The change in d_AB(t) is also accompanied by an increase of about 8° in the angle, Ω(t) (Fig 5D), between A, O, and B (O is the center of mass of residues Glu1264—Lys1426 in the β′ subunit) at t ≈ 24 μs, which coincides with the increase in d_AB(t) (Fig. 5C). Both d_AB(t) and Ω(t) decrease after DNA enters RNAP, which results in a tight grip on the dsDNA. Surprisingly, the distance d_OB(t) is almost constant (Fig. 5C). The totality of the results show that channel expansion occurs predominantly by swinging of the RS regions with the β′ subunit being stationary.

Fig. 5.

Channel opening mechanism in fast trajectories. (A) View of R·P_o. The view on the right is obtained by rotating the structure on the left by 90 ° about the vertical axis. The structural units that are close to downstream dsDNA are circled. The RS region and the promoter DNA are shown as ribbon diagrams. Two domains, Ala105-Ala499 and Glu1264-Lys1462 on the β′ subunit are displayed as spheres and colored magenta and pink, respectively. Residues on RS domain that make contact with the promoter DNA. Red spheres show that A, B, and O are centers of mass for RS domain, and the two selected domains of the β′ subunit. The distance between the closest residues on RS region and the β′ subunit (Pro244 and Ala122) is 14 Å, and the width of DNA is 22 Å. (B) The distance change between the closest points, residue Pro244 and Ala122 on the RS region and the β′ domain as a function of time averaged over ten fast trajectories. (C) Averaged distance changes d_AB (black) and d_OB (red) as a function of t. At t ≈ 19 μs, d_AB(t) increases, while d_OB(t) is relatively constant. (D) Time-dependent changes in Ω(t) = ∠AOB (see structure on top right in (A)) show a transient increase at t ≈ 19 μs. Results in (C) and (D) show that the RS domain (R in yellowish green and S in blue ribbons) swings away from the stationary β′ subunit (spheres in the structure on the right in (A)).

Conclusions

Using simulations we have shown that the formation of the transcription-competent R·P_o complex, which is known to occur through a series of intermediates (26, 27), proceeds in several steps and results in the transcription bubble formation. In fully accommodating the DNA, the internal dynamics of RNAP, which is hidden in many conventional experiments, plays a crucial role. Starting only with structural models of R·P_c and R·P_o, our work shows that efficient transcription bubble formation requires sequential promoter opening (beginning at around -10 and proceeding downstream), which is supported by experimental data (4, 28, 29). The simulations also make detailed testable quantitative predictions, which can advance our understanding of the dynamics of the promoter melting process.

The proposed three-step formation of the transcription bubble is similar to the time-resolved hydroxyl radical foot printing experiments (26, 30), which characterized the formation of the complex between the T7A1 promoter and the Escherichia coli RNA polymerase. From the time-dependent protection against hydroxyl radicals, they surmised that there are three global steps in the formation of R·P_o. In the first two stages the protection is attributed to interactions of melted DNA (template and nontemplate strands) with the RNAP, while the slower protection seen in the last stage is due to the entry of the dsDNA (+3 to +21) into RNAP. Our work suggests that the latter process is slow because it involves channel opening that is intimately coupled to the internal dynamics of the RNAP. However, a different study (27) suggested, using analysis of hydroxyl radical and potassium permanganate (KMnO₄) footprinting experiments, that the promoter (λ_RP) does not melt from -11 to +2 until after gaining entry into RNAP. The dynamical structural and energetic changes in our simulations do not support this picture. In particular, the stabilization of melted single-strand DNA around the -10 element by the aromatic residues of σ 2.3 could also prevent KMnO₄ from reacting with the intermediate (31). If such a mechanism prevails then the complex formation mechanism would be similar to the present and previous (26) studies. Finally, the predictions linking the internal RNAP dynamics and the bending of the promoter can be tested using single molecule experiments.

Methods

Self-Organized Polymer (SOP) Model for R·P_c → R·P_o Transition.

The large size of RNAP-DNA complex with 3,122 amino acids and a dsDNA with 150 nucleotides make it necessary to use a coarse-grained (32–35) models. Here we use a SOP model (14, 32, 34) for the enzyme and DNA. In the SOP model (32, 36), the structure of a protein is represented using only the C_α coordinates, with N^P being the number of amino acids). DNA is represented by the centers of mass of the nucleotides, with N^DNA being the number of nucleotides. The state-dependent energy functions for RNAP, DNA, and protein-DNA interaction are given in SI Text. The proposed SOP force field yields accurate value of the persistence length (44.3 nm) of the isolated DNA. In addition, the calculated B factors for the core RNAP are in excellent agreement with the measured data based on crystal structures except for a few solvent-exposed residues that are not involved in promoter melting. These results are predictions of the model and not a consequence of adjusting the parameters to obtain agreement with experiments. The accurate description of the properties of the isolated DNA and the core enzyme and previous predictions for forced-unfolding of GFP (37) and rigor to postrigor transition in Myosin V (38) provide ample validation of the coarse-grained model.

Brownian Dynamics Simulation of the R·P_c → R·P_o Transition.

The simulations of the R·P_c → R·P_o are based on the assumption that the local strain that triggers the open complex formation (due to DNA bending), propagates on a time scale that is faster across the structure than the time in which R·P_c → R·P_o transition occurs (32, 36, 39). The forces that trigger the R·P_c → R·P_o transition are computed from the energy function H(r_i|R·P_o), where the functional for H(r_i|R·P_o) is given by Eq. S1 in SI Text. The trajectories are generated by integrating the Langevin equations of motion for R·P_c → R·P_o transition (see SI Text for details).