Article pubs.acs.org/JPCB
Reversible-Switch Mechanism of the SAM-III Riboswitch Sha Gong,†,‡ Yujie Wang,† Zhen Wang,† Yanli Wang,† and Wenbing Zhang*,† †
Department of Physics, Wuhan University, Wuhan, Hubei 430072, P. R. China College of Mathematics and Physics, Huanggang Normal University, Huanggang, Hubei 438000, P. R. China
‡
ABSTRACT: Riboswitches are self-regulatory elements located at the 5′ untranslated region of certain mRNAs. The Enterococcus faecalis SAM-III (SMK) riboswitch regulates downstream gene expression through conformational change by sensing S-adenosylmethionine (SAM) at the translation level. Using the recently developed systematic helix-based computational method, we studied the co-transcriptional folding behavior of the SMK riboswitch and its shortened construct lacking the first six nucleotides. We find that there are no obvious misfolded structures formed during the transcription and refolding processes for this riboswitch. The full-length riboswitch quickly folds into the ON-state in the absence of SAM, and the coupling between transcription and translation is not required for the riboswitch to function. The potential to form helix P0 is necessary for the riboswitch to function as a switch. For this thermodynamically controlled reversible riboswitch, the fast helix-exchanging transition pathway between the two functional structures guaranteed that this riboswitch can act as a reversible riboswitch.
1. INTRODUCTION Riboswitches, as self-regulatory elements, are involved in regulating downstream gene expression, a function that has been traditionally attributed to proteins.1−5 Most riboswitches consist of two distinct domains: a conserved aptamer and a variable expression platform. 6−10 The aptamer, as an independent folding unit, is responsible for recognizing and binding the target metabolite, and the expression platform is involved in the genetic regulation.11−15 Riboswitches control gene expression through conformational changes triggered by ligand binding at the levels of translation, transcription, and splicing in cells.11,12,16−19 The Enterococcus faecalis SAM-III (SMK) riboswitch responds to the intracellular concentration of SAM or its derivative S-adenosylhomocysteine SAH to regulate the translation of the metK gene,20,21 which encodes the synthetase of SAM, a cofactor in methylation reactions of proteins, nucleic acids, and other biomolecules.22,23 The crystal structure of the SMK riboswitch with its ligand binding has been reported recently.24 This riboswitch was found to be a reversible switch in response to the ligand concentration in cells.25 Unlike most riboswitches, this riboswitch only has a simple architecture that utilizes a single domain for both ligand binding and gene regulation.21,26 Its Shine−Dalgarno (SD) sequence directly takes part in binding SAM and is sequestered by base pairing with the anti-SD (ASD) sequence in the presence of the ligand (Figure 1). Helices P1, P2, P3, and P4 form the binding pocket for SAM, and ligand binding stabilizes the OFF-state by introducing tertiary interactions. The sequestration of the SD sequence in the OFF-state inhibits initiation of the translation process and represses downstream gene expression.25,26 In the absence of SAM, the riboswitch © 2016 American Chemical Society
adopts an alternative folding pattern, at which the SD sequence is unstructured and free to bind the ribosomal subunit.27,28 Thus, the riboswitch acts as a genetic ON switch without the binding of the ligand. Previous studies have investigated many features of the SMK riboswitch such as ligand specificity and its structural basis,21 structure transition,2,28 and rates of ligand binding and dissociation.25,27 However, these studies have mainly focused on the full-length transcriptional products. In cells, folding of riboswitches, which are synthesized during the transcription process, follows a sequential progression.29,30 RNA folding pathways and kinetic traps during the transcription have been suggested to be different from that of the renaturation folding, and the transcription process could dictate RNA folding pathways and kinetic traps.31−33 For most riboswitches, their biological functions were found to be tied to the cotranscriptional folding.11,34−37 However, the co-transcriptional folding behaviors and the reversible switching mechanism of the SMK riboswitch remain unclear, for example, if transcription and translation are coupled for the riboswitch to function, then what is the transition pathway between the two function structures, and is the transition fast enough for the riboswitch to play the reversible switch function? To fully investigate regulation mechanisms of the SMK riboswitch, in this study we used the recently developed helix-based kinetic folding method to predict the co-transcriptional folding and refolding behavior of the riboswitch as well as its shortened construct without the Received: September 25, 2016 Revised: November 5, 2016 Published: November 8, 2016 12305
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conformation space is calculated using the nearest-neighbor model.38,39 The population kinetics, pi(t), for each state i at time t is described by the master equation dpi(t)/dt = ∑j[kj→i pj(t) − ki→j pi(t)], where ∑j denotes the sum over all the conformations, and ki→j and kj→i are the rate constants for the respective transitions. The above equation has a matrix form: dp(t)/dt = M·p(t), where p(t) denotes the vector of the population distribution, and M is the rate matrix with elements Mij = ki→j(i ≠ j) and Mii = −∑i≠j ki→j. For t > 0, the master equation yields the population kinetics p(t ) =
∑ Cmn m e−λ t m
m=1
(1)
where nm and −λm are the mth eigenvector and eigenvalue of rate matrix M, respectively, and coefficient Cm is determined by the initial conditions. The rate constants for formation (k+) and disruption (k−) of a stack are written as k+ = k0 e−TΔSstack/kBT and k− = k0 e−ΔHstack/kBT, where k0 is equal to 6.6 × 1012 s−1 for the formation/disruption of an AU base pair and 6.6 × 1013 s−1 for the formation/ disruption of a GC base pair,35,40−43 kB is the Boltzmann constant, and T is the temperature. The rates for formation and = k0 disruption of a loop-closing stack (and the loop) are kloop + e−(ΔSstack+ΔSloop)/kB and kloop = k0 e−ΔHstack/kBT, where −ΔSstack and − −ΔHstack are the entropy and enthalpy changes upon formation of the stack, respectively, and −ΔSloop is the entropy change of the loop. Recently, a helix-based kinetic model was proposed to predict the folding kinetics of long RNA sequences and had been used for a few sequences.40,41,43 In this model, the conformation space is constructed by using helices as the building blocks; the kinetic move is the addition or deletion of a helix or an exchange between two helices. If two states differ in one helix, the transition between them will be the formation and disruption of the helix. The most probable pathway for the helix formation is the zipping pathway, and the formation of the first stack is rate-limiting in the helix formation (Figure 2a). The zipping rate, kf, along the 1 → 2 → 3 pathway in Figure 2b can be calculated as43
Figure 1. Secondary structure model of the E. faecalis SMK riboswitch. The nucleotides within the paired region of helices P1, P2, P3, and P4 are colored differently: P1 (fuchsia), P2 (green), P3 (blue), and P4 (red). The SMK 84 construct lacks the first six nucleotides at the 5′ terminus (gray box).
first six nucleotides from the 5′ terminus of the RNA.32 We found that the riboswitch folded into the ON-state without obvious misfolded structures formed during both the transcription and the refolding processes. Although the transition between ON-state and OFF-state required a great conformational rearrangement, the fast helix-exchanging transition pathway guaranteed that it could function as a reversible switch in response to the ligand concentration.
2. THEORETICAL METHODS 2.1. Free-Folding Kinetics. For a given RNA chain, the conformation space is sampled by all possible secondary structures. The free energy of secondary structures in the
Figure 2. (a) Schematic free-energy landscape for a pathway from the open chain to a helix. (b) Formation of a helix along the zipping pathways. The first closing stack is denoted by the red circle. (c) Free-energy landscape for the transition between helices A and B. Solid line: the tunneling pathway. Dotted line: complete unfolding of helix A followed by refolding to helix B. The 3′ end of the RNA chain is denoted by purple color. 12306
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k f = k1 → 2K1(1 − K 2′K1′∑ (K 2′K1)n ) n=0
⎞ ⎛ 1 = k1 → 2K1⎜1 − K 2′K1′ ⎟ 1 − K 2′K1 ⎠ ⎝
(2)
where ki→j denotes the rate for the transition from state i to state j, and Ki and Ki′ are the forward and reverse probabilities, respectively k 2→3 k 2→1 , K1′ = , k 2→3 + k 2→1 + k 2→4 k 2→3 + k 2→1 + k 2→4 k3→5 + k3→6 k3→2 K2 = , K 2′ = k3→2 + k3→5 + k3→6 k3→2 + k3→5 + k3→6
K1 =
Figure 3. Four types of relationships between N-nt and (N + 1)-nt structures. The triangle and circle denote the last transcribed nucleotides at steps N and N + 1, respectively.
Rate kF for the helix formation is the sum of rates along all zipping pathways with different nucleation base pairs (Figure 2b). The rate for the helix deletion is calculated from the detailed balance condition, kU = kF e−ΔG/kBT, where ΔG is the folding free energy of the helix. If two helices A and B overlap with each other, they cannot coexist in the same structure. Compared with fully unfolding one helix followed by forming the other helix, the exchanging transition between the two helices occurs mostly through the tunneling pathway and has a much lower barrier (Figure 2c), wherein after one helix is partially disrupted, in each subsequent step, disruption of a stack in the helix is accompanied by the formation of a stack in the other helix. The rates between helices A and B for helix exchange can be estimated as43
beginning of step N + 1 can be summarized by the equations below32 P(N + 1)begin = P(N )end for a , b , and c ; P(N + 1)begin = 0 for d
For consecutive steps, the folding results of the previous step turn into the initial condition of the next step. Applying this method from the first step to the end of the transcription, we can compute the folding kinetics for the RNA chain during the transcription.
n
kA → B =
∏i ki n−1 j n ∑ j = 0 (∏i = 1 ki′∏m = j + 2 km)
kB → A = kA → B e−ΔGAB / kBT
3. RESULTS AND DISCUSSION 3.1. Co-transcriptional Folding and Refolding Behavior of the SMK Riboswitch. To investigate whether the action of the SMK riboswitch in cells depends on the transcription process, we first calculate the co-transcriptional folding behavior of the full-length SMK riboswitch without the ligand at a typical elongation rate of 50 nt/s. As the chain grows, the nascent RNA chain folds into a series of discrete intermediate states (Figure 4). Along the transcription, helix P5 (state C2) is formed as the first 19 nt are transcribed. Almost all riboswitches stay in this structure till the 36th nucleotide is transcribed, from which a new helix can be nucleated and the structure transits to C3. At the 44th step, the upper part of helix P3 is nucleated, and state C4, which consists of helices P5 and P3, quickly occupies most of the population. At about step 60, helix P0 becomes stable, and state C4 transits to the ON-state, which consists of helices P3, P5, and P0, by forming the nonlocal helix P0. At step 63, the ON-state is fully formed and occupies almost all of the population. From step 88, the repression stem P1 could be nucleated, and less than 2% of the population would transit from the ON-state to the OFF-state, as the ONstate with a free energy of −18.80 kcal/mol is more stable than the OFF-state (ΔGOFF = −17.40 kcal/mol). Therefore, in the absence of the ligand, the SMK riboswitch folds through a series of discrete intermediate states and finally populates at the ONstate with the SD sequence unstructured during the transcription process. This implies that it acts as a genetic ON switch to induce downstream gene expression without the ligand, in agreement with previous studies.25,26 The refolding kinetics of the full-length SAM-III riboswitch also shows that the thermodynamically favored ON-state is formed in less than 0.1 s, whereas the OFF-state only occupies a small proportion of the population (Figure 5). The results in
, (3)
where kn and k′n are the rate constants for the processes of formation (disruption) and disruption (formation) of a base stack in A (B), respectively. 2.2. Co-transcriptional Folding Kinetics. In the theory,32 the transcript of an L-nt RNA chain is divided into L transcriptional steps, each corresponding to the release of one nucleotide by RNA polymerase (RNAP) to freely form secondary structures. If the RNAP elongates the RNA chain with a speed of ν nucleotides/s, the (real) time window for each step will be 1/v s, that is, the polymerase spends 1/v s to synthesize a nucleotide. From time t when an N-nt chain is newly transcribed to time t + 1/v when the (N + 1)th nucleotide is (newly) transcribed, the N-nt chain samples the conformation space, and its population distribution is relaxed from [p1(N)begin, p2(N)begin, ..., pΩ(N)begin] to [p1(N)end, p2(N)end, ..., pΩ(N)end], where pi(N)begin and pi(N)end are the population of state i at the beginning and end of step N, respectively. At each such transcriptional step, the population kinetics is calculated in the same manner as the free-folding kinetics: first, the conformation space is generated, and the transition rates are calculated; then, the population relaxation within the folding time window is described by eq 1. It is important to note that the initial population at the current step is determined by the final population distribution at the previous step. Depending on possible structural changes upon elongation of the chain by one nucleotide, the structures are classified into four types (Figure 3). Then the population distribution at the 12307
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Figure 4. Population kinetics of main states (C1−C4) formed during the transcription of the SMK riboswitch without SAM at a transcription rate of 50 nt/s (a). (b) The transition pathways and structures formed during the transcription.
on the transcription process. Taken together, these results suggested, unlike the riboswitches operating at the transcription level,15,37 that the coupling between transcription and translation was not required for the SAM-III riboswitch to efficiently perform gene regulation. The ligand should bind the target to induce the function switch after the full-length chain is transcribed, and the flipping of the switch could be limited by ON-state and OFF-state transitions. 3.2. Helix P0 Is Crucial for the SMK Riboswitch To Perform Its Function. Previous study suggested that variable sequences outside the SAM-binding core critically influenced the conformational dynamics of the SAM-III riboswitch.28 The RNA construct lacking the first several nucleotides at the 5′ end of the RNA was found to favor a conformation resembling the genetic OFF-state even in the absence of SAM.27 To study the effect of these nucleotides on the function of the riboswitch, we predict the co-transcriptional folding behavior of an 84-nt SMK riboswitch construct lacking the first 6 nucleotides (Figure 6). It shows the same folding behavior as that of the full-length riboswitch before the 60th step (Figure 4), from which the fulllength RNA transits from structure C4 to the ON-state by forming helix P0. However, for the 84-nt construct, helix P0 is unable to form due to the lack of nucleotides. C4 occupies more than 98% of the population for a long transcription time, until the 72nd nucleotide is transcribed, when a new helix can
Figure 5. Refolding population kinetics of the 90-nt full-length SMK riboswitch.
Figures 4 and 5 showed that during both the free folding and the co-transcriptional folding processes, almost all the SAM-III riboswitches quickly folded into the thermodynamically favored ON-state. Hence, the regulation of its downstream gene translation by the structure of the riboswitch does not depend
Figure 6. Co-transcriptional folding kinetics of the 84-nt SMK riboswitch construct in the absence of SAM with 50 nt/s; population relaxation after the transcription is shown at the right side of the dotted line, where the end of step 84 is set as time 0 s. 12308
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transition from ON-state to OFF-state should be extremely slow through completely unfolding of helices P0 and P5, followed by refolding of helices P1, P2, and P4. The tunneling pathway, in which at first the helices of the ON-state are partially disrupted and, in each subsequent step, disruption of a base stack in the ON-state is accompanied by a formation of a base stack in the OFF-state, is much faster with a lower energy barrier.43 So the most probable transition pathway is the helixexchanging tunneling pathway (Figure 8a). According to eqs 2 and 3,43 the transition rate from ON-state to OFF-state and the reverse rate are 0.04 and 0.38 s−1, respectively, at 37 °C. The binding of the ligand to the full-length riboswitch can be described as a three-state transition model: ON-state ↔ OFFstate + SAM ↔ OFFb-state, where OFFb-state is the OFF-state with ligand bound. Ligand binding can stabilize the ligandbound OFF-state by ΔGbinding = kBT ln(kon[SAM]/koff) kcal/ mol, where [SAM] is the concentration of SAM, and kon and koff are the association and disassociation rates, respectively, which have been experimentally measured for the full-length riboswitch as kon = 0.11 μM−1 s−1 and koff = 0.089 s−1.25 The transition rate from OFF-state to OFFb-state was assumed to be the effective binding rate, keff = kon[SAM], and the transition rate from OFFb-state to OFF-state is the dissociation rate, koff. Assuming the initial state is the ON-state, most riboswitches could transit to the OFFb-state within 100 s in the presence of 500 μM SAM (Figure 8b), which is in agreement with the experimental measured value of 1 min.28 When the ligand concentration decreases to 1 μM, more than half of the riboswitch would transit from the genetic OFF-state (OFFbstate) to the genetic ON-state (ON-state) within 60 s (Figure 8c). These results suggest that the SMK riboswitch can switch back and forth to regulate translation of its host gene quickly in response to the ligand concentration. The major time scales that control the function of the SMK riboswitch are (1) the ligand-binding rate, kon = 0.11 μM−1 s−1, (2) the dissociation rate, koff = 0.089 s−1, and (3) the mRNA degradation rate, kmRNA = 3 min−1.47 A thermodynamic regime is that the time needed to attain an RNA−ligand equilibrium is short compared with the transcriptional time scale. The switching rate for the SMK riboswitch is faster than the degradation rate of the riboswitch, so the functional status of the SMK riboswitch at any given time is governed by thermodynamic principles.
be formed and the population of structure C4 begins to transit to structure C5, which does not form for the full-length riboswitch (Figure 4). As the 77th nucleotide is free to form structure, structure C5 begins to transit to the OFF-state. At the end of the transcription, structure C5 and the OFF-state occupy about 52.5 and 45.1% of the population, respectively. The free energy of structure C5 and the OFF-state are −16.32 and −17.40 kcal/mol, respectively, so they do not reach equilibrium at the end of transcription. Another 0.5 s are required to significantly reach equilibrium, and then the OFFstate occupies about 80% of the population. Although the ribosome-binding time is not well known, the time required to transcribe the mRNA footprint for the ribosome can provide a lower bound.44 Crystal structures and footprinting studies suggest that this involves another 15-nt downstream of the 3′ end of the riboswitch expression platform,45,46 requiring about 0.3 s at typical transcription rates. During the transcription of the 84-nt RNA construct, the ON-state is not formed due to the lack of the nucleotides at the left shoulder of helix P0. The refolding process of the 84-nt RNA also shows that structure C5 as an intermediate can be formed, but it is quickly replaced by the OFF-state (Figure 7), and the thermodynami-
Figure 7. Refolding population kinetics of the 84-nt SMK riboswitch.
4. CONCLUSIONS Riboswitches, as typical functional mRNAs, which are synthesized during the transcription process, adopt alternative folds to control downstream gene expression.48−50 It has been found that for many riboswitches, their functions are sensitive to co-transcriptional folding events.15,29,35,37 To understand the functions of riboswitches, it is important and necessary to investigate their co-transcriptional folding behaviors. However, sequential folding during the transcription of mRNAs poses a serious challenge in detecting these structures as they are formed within a short time.30,51 Using the recently developed systematic helix-based computational method to predict the co-transcriptional folding kinetics,32 we theoretically studied the co-transcriptional folding behavior and refolding kinetics of the E. faecalis SAM-III riboswitch. The riboswitch, with a simple architecture to bind the ligand and regulate gene expression, displays many unique features in its regulation. Both the refolding and the cotranscriptional folding kinetics of the full-length riboswitch showed that most RNAs predominantly folded into the ON-
cally favored OFF-state can be formed in less than 1 s. This implies that most riboswitches fold into the OFF-state before the ribosome can bind to the riboswitch. Thus, the 84-nt SMK riboswitch shortened construct functions as a “constitutiveOFF” switch to repress downstream gene expression even in the absence of SAM in cells, consistent with experimental observation.28 In other words, the potential to form helix P0 is crucial for the SMK riboswitch to perform its switch function. 3.3. ON−OFF-State Transition Is through a HelixExchanging Pathway. The free-folding kinetics and the cotranscriptional folding kinetics indicated that without SAM, the SMK riboswitch would predominantly fold to the thermodynamically favored ON-state. The OFF-state was suggested to be a SAM-binding-competent structure.28 But the transition from ON-state to OFF-state needs to undergo enormous conformational rearrangements, as it needs to break helices P0 and P5 and form helices P1, P2, and P4. What is the transition pathway, and is the transition rate fast enough for this reversible riboswitch to function? Helices P0 and P5 of the ON-state overlap with those of P1, P2, P4 of the OFF-state. The 12309
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Figure 8. (a) Exchanging transition pathway between the ON-state and the OFF-state and the schematic free-energy landscape of the pathway (unit kcal/mol). (b) Transition from ON-state to OFFb-state through the pathway: ON ↔ OFF + SAM ↔ OFFb-state in the presence of 500 μM SAM. (c) Transition from OFFb-state to ON-state when the ligand concentration decreases to 1 μM.
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ACKNOWLEDGMENTS This work was partly supported by the National Natural Science Foundation of China under grant nos. 31270761 and 1157324 (W.Z.).
state. This implies that the function of the riboswitch is not tied to the transcription process and that the ligand binding occurs after the transcription. Without ligand, the ON-state is the most stable state. Upon SAM binding, the equilibrium is shifted toward the thermodynamically favorable ligand-bound OFFstate with the repression stem formed. The SMK riboswitch exerts its function by thermodynamic control, and its switch efficiency is linked to the stability of the two function structures instead of the transcription context. Although the ON−OFF transition requires enormous conformation rearrangements, the fast helix-exchanging transition pathway between the two function structures allows it to perform as a reversible switch in response to the ligand concentration. The shortened or mutated RNA constructs, which disrupt helix P0 of the ON-state, are not capable of switching and behave as a constitutive-OFF switch. These results demonstrated that the riboswitch is highly evolved and has selected sequences. However, the current method has limitations. The tertiary interactions and ion effects,52,53 which may alter the folding pathways, are neglected. Future development of the model would incorporate these effects into the model.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Wenbing Zhang: 0000-0001-6880-7087 Notes
The authors declare no competing financial interest. 12310
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DOI: 10.1021/acs.jpcb.6b09698 J. Phys. Chem. B 2016, 120, 12305−12311
