In Situ Monitoring of Structural Changes during Formation of 30S

May 5, 2014 - We performed an energy dissipation measurement by using a quartz-crystal microbalance-admittance (QCM-A) technique for in situ monitorin...
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In Situ Monitoring of Structural Changes during Formation of 30S Translation Initiation Complex by Energy Dissipation Measurement Using 27-MHz Quartz-Crystal Microbalance Hiroyuki Furusawa,*,†,‡ Yumi Tsuyuki,‡ Shuntaro Takahashi,‡,§ and Yoshio Okahata*,†,‡ †

Innovative Flex Course for Frontier Organic Material Systems (iFront), Yamagata University, 4-3-16 Jonan, Yonezawa, Yamagata 992-8510, Japan ‡ Department of Biomolecular Engineering, Tokyo Institute of Technology, 4259, Nagatsuda, Midori-ku, Yokohama, Kanagawa 226-8501, Japan § Frontier Institute for Biomolecular Engineering Research (FIBER), Konan University, 7-1-20 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan S Supporting Information *

ABSTRACT: Ribosome is a bionanomachine that facilitates an orderly translation process during protein synthesis in living cells. Real-time monitoring of conformational changes in ribosomal subunits in aqueous solution is important to understand the regulatory mechanism of protein synthesis, because conformational changes in ribosome in E. coli have been predicted to operate the switch from translation initiation to an elongation process during translation. We performed an energy dissipation measurement by using a quartz-crystal microbalance-admittance (QCM-A) technique for in situ monitoring of conformational changes in pre-30S translation initiation complex in response to the binding of fMet-tRNAfMet in aqueous solution. The addition of fMet-tRNAfMet caused changes in the physical property (increased dehydration and elasticity) in 30S ribosomal subunit in the presence of mRNA and IF2/guanosine 5'-triphosphate (GTP) on the QCM plate. Furthermore, two sequential changes triggered by the addition of fMet-tRNAfMet were observed in 30S ribosomal subunit bound to mRNA in the presence of IF2/GTP and IF3. These observations suggest that the structural changes in 30S ribosomal subunit caused by the binding of fMet-tRNAfMet with IF2/GTP in the presence of IF3 could act as a switch to regulate the orderly processing in the construction of translation initiation complex, because the structural distinction can be a guidepost in the process for the relevant biomolecules.

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frame for decoding of mRNA. Therefore, the initiator tRNA must be precisely positioned on the ribosome associated with the mRNA. The initiation process is controlled by three translation initiation factors, called IF1, IF2, and IF3. Roles of each initiation factor have been previously reported (see Figure 1).2,5−10 First, 70S ribosome dissociates into 50S and 30S ribosomal subunits, followed by the binding of IF1 and IF3 to 30S ribosomal subunit. The A-site is blocked by IF1 from binding with other tRNAs. Then, mRNA, fMet-tRNAfMet, and guanosine 5'-triphosphate (GTP)-bound IF2 (IF2/GTP) bind to the 30S subunit to form the 30S initiation complex (30S-IC),

ibosome is a bionanomachine used for protein synthesis, which is facilitated by a precise translation process. The translation process is divided into three steps: translation initiation, elongation, and termination.1 In particular, translation initiation is one of the key steps in protein synthesis that occurs in the bacterial ribosome, which is the rate-limiting step in protein synthesis.2,3 In this process, the ribosome assembles on the mRNA with the help of some factors in Escherichia coli.1−4 First, a small 30S ribosomal subunit associates with mRNA mediated by interactions between Shine-Dalgarno (SD) sequence in the ribosome-binding site on mRNA and the 16SrRNA in the 30S ribosomal subunit. Second, the initiator tRNA that is referred to as fMet-tRNAfMet enters the P-site in the ribosome and interacts with mRNA by base-pairing between the anticodon of the initiator tRNA and the start codon of mRNA.5 This assembly results in the setting-up of the reading © 2014 American Chemical Society

Received: February 4, 2014 Accepted: May 5, 2014 Published: May 5, 2014 5406

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Figure 1. General scheme of the translation initiation process for the protein synthesis in bacteria. First, 70S ribosome splits into 50S and 30S ribosomal subunits and then 30S subunit interacts with mRNA, IF1, IF2/GTP, IF3, and fMet-tRNAfMet to form the 30S initiation complex (30S-IC). Next, 50S subunit reassociates to form pre-70S-IC, accompanying release of IF1 and IF3. Finally, IF2 dissociates from the ribosome after the hydrolysis of GTP to GDP by GTPase activity on 50S subunit, resulting in the formation of the 70S initiation complex (70S-IC).

where IF3 helps to ensure accuracy of the fMet-tRNAfMet setting. Next, the 50S subunit reassociates with 30S-IC, accompanying release of IF1 and IF3. Subsequently, GTP hydrolysis occurs and IF2/GDP dissociates from pre-70S-IC. Finally, the 70S initiation complex (70S-IC) is formed; it is composed of 70S ribosome, mRNA, and fMet-tRNAfMet. After the completion of this process, the ribosome proceeds to elongate the polypeptide chain. In this process, the trigger for the association of 50S ribosome subunit, a key step in pre-70SIC formation, could probably be the conformational changes in 30S ribosomal subunit caused by base-pairing between the anticodon of fMet-tRNAfMet and the start codon of the mRNA. However, the dynamic mechanism associated with the initiation of translation is still unclear due to a paucity of structural information in real-time. Recently, crystallographic studies on several initiation complexes have made significant progress in understanding the structural and mechanistic aspects of protein synthesis.1,2,11 For example, X-ray structures of several ribosome complexes modeling translation initiation were compared, and the movement of the SD sequence on mRNA and the anti-SD sequence of 16S-rRNA during the initiation process was visualized.2,12 Moreover, cryo-electron microscopy (cryo-EM) has provided important insights into the reconstruction of ribosomes and protein synthesis factors.1,2,11 For example, direct visualization of 30S initiation complex containing mRNA, fMet-tRNAfMet, IF1, and IF2/GTP and different conformations according to different functional states have been reported.2,13 These reports implicate potential changes in the structural property of the complex during the translation initiation process. Nevertheless, the information obtained by crystallography and cryo-EM methods represents random static snapshots that were not obtained from a real-time observation in an aqueous solution. A piezoelectric quartz-crystal microbalance (QCM) driven by an oscillation circuit is a highly sensitive mass measuring device, whose fundamental frequency decreases linearly upon an

increase in mass on a 27-MHz QCM electrode at a nanogram level.14−16 Therefore, the QCM technique has been applied as a biosensor in various biomolecular interaction analyses.15−23 In the case of hydrophilic biomolecules, a frequency change includes the changes in bound and vibrating water mass together with the loading mass, and the energy dissipation of QCM oscillation is related to the amount of hydration and viscoelastic properties of loading materials.24−26 When the quartz-crystal oscillator is connected with a network-analyzer to determine the crystal admittance, the resonance frequency F and the energy dissipation factor D, corresponding to an inverse of the frequency quality Q, can be obtained from conductance-frequency plots (QCM-admittance method [QCM-A]), in which F is the maximum conductance point and D is the ratio of the difference between two frequencies at half the height of the maximum conductance (F2 − F1) to the resonance frequency F (Figure 2).25,27 Thus, in measurements using QCM-A, loading of a mass on the QCM plate results in a frequency shift (ΔF) to the lower frequency and the increase of the viscoelastic property of the material on the QCM plate results in the broadening of the resonant curve, indicating an increase in energy dissipation (D). Therefore, we can monitor the changes in the physical property of various biomolecules immobilized on a QCM-A plate by measurement of both ΔF and ΔD values. We have reported a real-time monitoring of the conformational changes in calmodulin due to Ca2+ ion-binding by using the changes in the amount of hydration and viscoelasticity by determining the frequency shifts (ΔF) and energy dissipation changes (ΔD) successfully.27 In this paper, we report in situ monitoring of structural changes during the formation of 30S translation initiation complex (30S-IC) by using the frequency and energy dissipation measurements (ΔF and ΔD values) of QCM-A, after the addition of fMet-tRNAfMet and initiation factors to 30S ribosomal subunit bound to biotinylated mRNA, which was immobilized on NeutrAvidin-modified QCM plate (Figure 2B). The decrease in the energy dissipation (the elasticity increase) 5407

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Figure 2. Schematic illustrations of (A) a 27-MHz QCM system connected with a vector network analyzer for an admittance analysis of a quartzcrystal oscillator (QCM-A system) and (B) in situ measurement of resonance frequencies (F) and energy dissipation (D) calculated from resonance curves of the oscillator obtained by using frequency sweep on the QCM-A system, depending on the binding of fMet-tRNAfMet to 30S subunit bound on the biotinylated mRNA on the NeutrAvidin-modified QCM plate. A frequency shift (ΔF) and a change in energy dissipation (ΔD) were calculated using the represented equations, as described previously.25,27

aminoacyl-tRNA synthetases (Met-RS and Lys-RS) were prepared using the system for expression of recombinant proteins in E. coli and purified by column-chromatography as described previously.20,29−31 Two tRNAs (tRNAfMet and tRNALys) were prepared from each of the overexpressed strains and purified by anion exchange chromatography as described previously.19,28,32 fMet-tRNAfMet was prepared enzymatically through aminoacylation of tRNAfMet with methionine by methionyl-tRNA synthetase (Met-RS) and then formylation by methionyl tRNA formyltransferase (MTF).19,28,31 Similarly, Lys-tRNALys was aminoacylated with lysine by lysyl-tRNA synthetase (Lys-RS). These samples were stored at −80 °C until they were used. Biotinylated mRNA. As previously reported, the template DNA for transcription to mRNA was designed to include SD sequence (AGGAGG) and the start codon (AUG) in 76 nt sequence (5′-GGGAGAUUCCCCAUGAUAACAUGGAUUCACAGGAGGCACAAUACAUGAAAAACUAGUCCGGGUACCGAGCUCGAAUU-3′).19 The template DNA with T7 promoter sequence at the 5′-side was inserted into pUC18

of 30S ribosomal subunit bound on mRNA was observed, in response to the binding of both fMet-tRNAfMet and IF2/GTP. These results suggest that the conformational change into the more elastic structure of 30S-IC, meaning a rigid state, could be triggered by the binding of fMet-tRNAfMet to the surface of 30S ribosomal subunit.

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EXPERIMENTAL SECTION Materials. EZ-Link Biotin-LC-Hydrazide and 1-ethyl-3-[3(dimethylamino)propyl]carbodiimide (EDC) were purchased from Takara Bio Inc. (Shiga, Japan) and Dojindo Laboratories (Kumamoto, Japan), respectively. NeutrAvidin was purchased from Pierce Co. (Tokyo, Japan). N-Hydroxysuccinimide (NHS) and kanamycin were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Unless otherwise noted, other reagents were purchased from Nacalai Tesque Co. (Kyoto, Japan) and used without further purification. Factors for Cell-Free Translation Initiation. E. coli 30S ribosomal subunits were prepared and purified as described previously.19,28,29 Initiation factors (IF1, IF2, and IF3) and two 5408

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Figure 3. Typical time course of frequency changes (ΔFwater) and energy dissipation changes (ΔDwater), responding to additions of (A) the mixture solution of 30S subunit (8 nM) and IF2/GTP (50 nM) and, then, (B) the solution of fMet-tRNAfMet solution (500 nM) into the mRNAimmobilized 27-MHz QCM cell filled with the buffer solution (10 mM HEPES-KOH, pH 7.6, 5 mM MgCl2, 0.5 mM CaCl2, 1 mM GTP, 0.1 mg mL−1 BSA, 200 nM IF2/GTP at 25 °C). (C) The enlarged view of (B), where curves a and b are ΔFwater and ΔDwater in the presence of kanamycin (100 μM) under similar conditions.

plasmid and then digested by EcoRI. The mRNA was prepared by a runoff transcription method using CUGA7 RNA polymerase (Nippon Gene, Co., Tokyo, Japan). After purification, the 3′-end of mRNA was oxidized by NaIO4 and reacted with EZ-Link Biotin-LC-Hydrazide. After reducing with the addition of NaBH4, the biotinylated mRNA was purified by using Microspin G-25 Columns (GE Healthcare, Co., Tokyo, Japan) and stored at −80 °C until further use. Analysis of 27-MHz QCM-Admittance (QCM-A). The analysis of the admittance of a 27-MHz piezoelectric quartz crystal was performed as described in previous papers.25,27 AFFINIX Q4 (Initium, Inc., Tokyo, Japan) connected with a

vector network analyzer (model: R3754B, Advantest, Co. Ltd., Tokyo, Japan) through a π-network (Sansei Denshi Co. Ltd., Tokyo, Japan) was used as a QCM-A apparatus. The QCM instrument had four 500 μL cells that were equipped with a 27MHz QCM plate (8.7 mm diameter of an AT-cut shear-mode quartz plate and an area of 5.7 mm2 of an Au electrode) at the bottom of the cell along with a stirring bar and a temperature controlling system. The admittance curve was obtained using a linear frequency sweep with 201 data points in frequency spans of 40 kHz around the 27-MHz resonance frequency region for measurements. Each admittance curve was treated with 16 time averages. One F and one D point could be obtained at each 1 s. 5409

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ssDNA.25 The coverage of the immobilized mRNA was calculated to be 0.6% with 1.6 nm2 molec.−1 of the mRNA area. The distance between mRNAs on the QCM plate was calculated to be approximately 18 nm. This distance is adequate for the access of 30S ribosomal subunit (maximum diameter: 18−20 nm) to mRNA. Measurement of Frequency Changes (ΔFwater) and Energy Dissipations (ΔDwater). The mRNA-immobilized QCM cell was filled with 500 μL of HEPES buffer solution (10 mM HEPES-KOH, pH 7.6, 5 mM MgCl2, 0.5 mM CaCl2 at 25 °C) including 1 mM GTP, 0.1 mg mL−1 BSA, and 200 nM IF2/GTP. After the resonance frequency (Fwater) and the energy dissipation value (Dwater) reached the steady state, the mixture solution of 30S ribosomal subunit and IF2/GTP was preincubated at 37 °C for 5 min and then injected at the final concentration of 8 nM of 30S ribosomal subunit and 50 nM of IF2/GTP. ΔFwater and ΔDwater values were recorded over time to monitor the response to the addition of fMet-tRNAfMet solution into the cell. The solution was vigorously stirred to avoid any effect of slow diffusion of the added molecules in the cell.

An exponential moving average analysis was applied to the raw data to reduce noise levels. The frequency noise was less than ±2 Hz, and the dissipation noise was less than ±0.1 × 10−6 in the buffer solution. Calibration of 27-MHz QCM-A. Sauerbrey’s equation (eq 1) is employed on an AT-cut shear mode QCM in the air phase: ΔFair = −

2F0 2 Δm A ρq μq

(1)

where ΔFair is the measured frequency change in the air phase (Hz), F0 is the fundamental frequency of the quartz crystal (27 × 106 Hz), Δm is the mass change (g), A is the electrode area (0.057 cm2), ρq is the density of quartz (2.65 g cm−3), and μq is the shear modulus of quartz (2.95 × 1011 dyn cm−2). In the air phase, a 0.62 ng cm−2 mass increase for each 1 Hz decrease in frequency was obtained in previous experiments.17−23,25−27 This agreed well with the value of 0.61 ng cm−2 Hz−1 calculated numerically from eq 1. The frequency change in the water phase (ΔFwater) is represented by eq 2: ΔFwater

2F0 2 ΔF = − water Δm ΔFair A ρq μq

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RESULTS AND DISCUSSION Binding of 30S Subunit and IF2/GTP onto mRNA. Figure 3A shows a typical change in frequency (ΔFwater) and a change in energy dissipation (ΔDwater) of the mRNAimmobilized QCM in aqueous solution, in response to the addition of the mixture solution of 30S subunit and IF2/GTP. The decrease in the frequency indicates the mass increase due to the binding of 30S subunit and IF2/GTP on mRNA. On the contrary, decreases in the frequency in the absence of mRNAimmobilized QCM were hardly observed as nonspecific binding onto the NeutrAvidin surface (date not shown). The ΔFwater = −1200 Hz of 30S subunit-binding amount was estimated to be 233 ng cm−2 (0.23 pmol cm−2) by using the calibration of 0.2 ng cm−2 Hz−1 as 30S subunit.26 This value indicates the binding of one 30S subunit per 2.6 mRNAs by considering the amount of mRNA immobilized up to ΔFwater = −100 Hz on the QCM, corresponding to 15 ng cm−2 (0.6 pmol cm−2). Because the ΔFwater/ΔFair value for 30S ribosomal subunit is 3.2,26 we should note that the value of ΔFair (dry mass) for 30S ribosomal subunit bound on mRNA could be calculated to be −375 Hz. Thus, −825 Hz subtraction of ΔFwater and ΔFair should correspond to the amount of hydrodynamic water vibrating with 30S subunit on the oscillating QCM surface.25 When the 30S subunit and IF2/GTP were bound onto mRNA, the energy dissipation value (ΔDwater) was increased. This indicates that the energy dissipation occurred by the hydrated and/or viscous structure of the complex on the oscillating QCM surface. The value of the energy dissipation per unit mass [ΔDwater/(−ΔFair)], which reflects the intrinsic viscoelastic properties, was calculated to be 5.1 × 10−8 Hz−1. This value was larger than those of globular proteins such as NeutrAvidin and BSA (1.4 × 10−8 and 1.6 × 10−8 Hz−1, respectively) and was smaller than those of linear biomolecules such as ssDNA (55 nt) and denatured BSA (11 × 10−8 and 8.3 × 10−8 Hz−1, respectively), showing that the 30S subunit on mRNA is considerably soft or flexible compared with NeutrAvidin and native BSA.25 Binding of fMet-tRNAfMet onto the Complex of 30S Subunit, IF2/GTP, and mRNA. After the formation of the complex of 30S subunit and IF2/GTP on mRNA, 500 nM of fMet-tRNAfMet was sequentially added in the QCM cell (Figure

(2)

where a correction factor, ΔFwater/ΔFair, is inserted into eq 1 in order to consider the effects of hydration and/or viscoelasticity of substances on the QCM.25,26 ΔFwater/ΔFair values indicate the hydrodynamic water (bound water and vibrated water) ratio per unit mass. We reported previously that ΔFwater/ΔFair values would depend on the molecular structure such as hydrodynamic radius (γH) of substances25,26 and the ratio of the hydrophilicity/hydrophobicity of substances.27 We obtained the ΔFwater/ΔFair value for 30S ribosomal subunit bound onto the QCM plate.26 There was a good linear correlation between ΔFwater and ΔFair, with a slope (= ΔFwater/ΔFair) of 3.2 ± 0.1 for the binding of 30S ribosomal subunit on the QCM plate.26 Thus, the frequency decrease in water (ΔFwater) due to the binding of 30S ribosomal subunit was 3.2 times larger than that in the air phase, owing to the vibrations of the hydrating water along with large ribosomes. In addition, the value of energy dissipation per unit mass, ΔDwater/(−ΔFair) could reflect the intrinsic viscoelastic properties of molecules.25,27 These parameters are useful to characterize the state of biomolecules in aqueous solutions. Immobilization of mRNA on the QCM Plate. NeutrAvidin (60 kDa) was covalently immobilized on the Au electrode on the QCM plate as described previously.17,19,27 Briefly, to the cleaned bare Au-electrode, 3,3′-dithiodipropionic acid was immobilized, and then, carboxylic acids were activated as N-hydroxysuccinimidyl esters on the surface. NeutrAvidin was reacted with activated esters by mounting the aqueous solution on the QCM plate. The binding behaviors of biotinylated mRNA (25 kDa) to the NeutrAvidin-immobilized QCM were followed in HEPES buffer solution (10 mM HEPES-KOH, pH 7.6, 5 mM MgCl2, 0.5 mM CaCl2 at 25 °C), and ΔFwater values were measured over a time period. When the ΔFwater value corresponding to the amount of anchored-mRNA reached a predetermined value (−100 Hz), 5 μM of free biotin was added to regulate the immobilization amount. The −100 Hz of the amount of mRNA was estimated to be 15 ng cm−2 (0.6 pmol cm−2) using the calibration of 0.15 ng cm−2 Hz−1 as 5410

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Figure 4. Typical time course of frequency changes (ΔFwater) and energy dissipation changes (ΔDwater), responding to additions of (A) Lys-tRNALys (500 nM) onto the complex of 30S subunit, IF2/GTP, and mRNA, (B) fMet-tRNAfMet (500 nM) onto the complex of 30S subunit and mRNA in the absence of IF2/GTP, and (C) fMet-tRNAfMet (500 nM) onto the mRNA immobilized on the QCM in the absence of 30S subunit (10 mM HEPES-KOH, pH 7.6, 5 mM MgCl2, 0.5 mM CaCl2, 1 mM GTP, 0.1 mg mL−1 BSA, 8 nM 30S subunit, and 250 nM IF2/GTP at 25 °C).

3B). Although the decreases in ΔFwater (the mass increases) due to the binding of fMet-tRNAfMet (Mw: 25 kDa) were expected to be around −30 Hz, the ΔFwater increased (the mass decreased) to around 220 Hz. Moreover, the ΔDwater decreased (the elasticity increased) synchronously (see also Figure 3C). However, the frequency and the energy dissipation hardly changed in the presence of 100 μM kanamycin, which is one of the aminoglycoside antibiotics that inhibits fMet-tRNAfMet binding to IF2/GTP due to the ability of its interaction with RNA.33 IF2/GTP has been reported to facilitate the association of fMet-tRNAfMet with mRNA on 30S subunit by the bridge interaction between fMet-tRNAfMet and 30S subunit.2,13 We consider that the binding equilibrium of IF2/GTP to 30S subunit could be very fast and IF2/GTP could bind tightly to 30S subunit together with fMet-tRNAfMet. Thus, these results indicate that the observed ΔFwater and ΔDwater reflect the changes in the physical properties of the complex of 30S subunit and IF2/GTP on the QCM plate triggered by the fMettRNAfMet binding through the IF2/GTP−fMet-tRNAfMet

interaction. The elasticity increase in 30S subunit is reasonable, as clamping with IF2/GTP−fMet-tRNAfMet on 30S subunit is like a tense arch observed by cryo-EM.13 To rule out the possibility that the increase in ΔFwater and the decrease in ΔDwater in response to the addition of fMettRNAfMet could be caused by the desorption of the bound 30S subunit from mRNA on the QCM plate, we also observed ΔFwater and ΔDwater of the QCM, where the 30S subunit was directly immobilized physically on the surface, in response to the addition of fMet-tRNAfMet in the presence of mRNA and IF2/GTP. We obtained similar results to that obtained on the mRNA-immobilized QCM (Supporting Information, Figure S1A). Therefore, we excluded the possibility of desorption of the bound 30S subunit from the QCM surface. The observed frequency changes (ΔFwater) and energy dissipation changes (ΔDwater) illustrated in Figure 3C are similar to ΔFwater and ΔDwater of the Calmodulin (CaM)immobilized QCM in response to the addition of Ca2+ ions.27 In the case of Ca2+ ions binding to CaM, the results were 5411

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Figure 5. Typical time course of frequency changes (ΔFwater) and energy dissipation changes (ΔDwater), responding to the addition of fMet-tRNAfMet (500 nM) to the complex of 30S subunit, IF2/GTP, and mRNA in the presence of (A) IF1 (250 nM), (B) IF3 (250 nM), and (C) both IF1 and IF3 (each 250 nM) (10 mM HEPES-KOH, pH 7.6, 5 mM MgCl2, 0.5 mM CaCl2, 1 mM GTP, 0.1 mg mL−1 BSA, 8 nM 30S subunit, and 250 nM IF2/ GTP at 25 °C).

explained by conformational changes of CaM: the ΔFwater increase (the apparent mass decrease) corresponded to the dehydration caused by the formation of a more open conformation that exposes more hydrophobic surface, and the ΔDwater decrease (the elasticity increase) corresponded to the conformational change from a flexible CaM state involving random coil elements within each globular domain to a more rigid dumbbell-shaped Ca2+/CaM conformation. As shown in Figure 3B,C, the large increase in ΔFwater (18% of apparent mass decrease) and decrease in ΔDwater (16% of apparent elasticity increase) observed when fMet-tRNAfMet bound to the complex of 30S subunit and IF2/GTP on mRNA could be mainly attributed to the conformational change of 30S subunit, because the mass of the 30S subunit (900 kDa) is extremely large compared with IF2/GTP (96 kDa) and fMet-tRNAfMet (25 kDa). Thus, we consider that the viscoelastic and hydrated complex of 30S subunit and IF2/GTP became more elastic, meaning “rigid”, and more dehydrated by the binding of fMet-tRNAfMet along with the conformational changes (see also Figure 2B).

Effect of fMet-tRNAfMet and IF2/GTP on Conformational Changes in 30S Subunit. IF2/GTP recognizes a charged fMet-tRNAfMet by the C-terminal domain and plays a role in the stabilization of fMet-tRNAfMet on the P-site of 30S ribosomal subunit (see Figure 1). When Lys-tRNALys was injected to the complex of 30S subunit and IF2/GTP on mRNA instead of fMet-tRNAfMet, very little ΔFwater and ΔDwater were observed (Figure 4A). This suggests that the codonanticodon interaction or the IF2/GTP−tRNA interaction is important to cause the conformational change in the complex. When fMet-tRNAfMet was injected to the 30S subunit on mRNA in the absence of IF2/GTP or 30S subunit, no ΔFwater and ΔDwater changes were observed (Figure 4B,C), suggesting that the conformational change in 30S subunit require both 30S subunit and IF2/GTP with fMet-tRNAfMet , which are components of a pre-70S-IC (see Figure 1). Therefore, conformational change has the potential to act as a switch to the next step in pre-70S-IC formation. Effect of Other Initiation Factors (IF1 and IF3) on Conformational Changes of 30S Subunit. When fMettRNAfMet was injected to the complex of 30S subunit, IF2/ 5412

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Figure 6. Typical time course of frequency changes (ΔFwater) and energy dissipation changes (ΔDwater), responding to additions of (A) IF3 (250 nM) and then (B) fMet-tRNAfMet (500 nM) to the complex of 30S subunit, IF2/GTP, and mRNA on the QCM (10 mM HEPES-KOH, pH 7.6, 5 mM MgCl2, 0.5 mM CaCl2, 1 mM GTP, 0.1 mg mL−1 BSA, 8 nM 30S subunit, and 250 nM IF2/GTP at 25 °C).

GTP, and mRNA in the presence of IF1, the large increase in ΔFwater (apparent mass decrease due to the dehydration) and the small decrease in ΔDwater (the elasticity increase) were observed (Figure 5A), in comparison to those in the absence of IF1 (Figure 3C). This suggests that IF1 was involved in the conformational change of 30S subunit, resulting in the larger dehydration. The change could correspond to a better positioning of the mRNA on 30S subunit due to IF1.9 On the other hand, in the presence of IF3, the ΔFwater increased slightly at first and then decreased shortly (Figure 5B), indicating that two sequential changes should occur on the complex-immobilized QCM. Because the state change after 30S-IC formation could be required to promote reassociation of 50S subunit in the pre-70S-IC formation (Figure 1), we surmise that the conformational changes in 30S subunit triggered by IF2/GTP-fMet-tRNAfMet in the presence of IF3 could induce the ready-state to associate with 50S subunit. The initial increase in ΔFwater was probably due to the conformational change in 30S subunit (the dehydration) caused by the binding of fMet-tRNAfMet, and the subsequent decrease in ΔFwater (the apparent mass increase) was probably facilitated by the additional hydration due to the structural change in 30S subunit affected by association of IF3. The increase in ΔDwater (the viscosity increase) illustrated in Figure 5B corresponds to the subsequent decrease in ΔFwater because the time course of ΔDwater is consistent with that of the second decrease of ΔFwater. This suggests that the subsequent change in 30S subunit depending on association of IF3 would be accompanied by the change in the viscosity of 30S subunit. Thus, it is expected that the physical change in 30S subunit could act as a switch to

associate with 50S subunit. In addition, we have observed the decrease of IF3-affinity to 30S-IC responding to the addition of fMet-tRNAfMet in our previous experiments using a QCM method elsewhere.34 The lower affinity of IF3 to 30S subunit could be caused by the changes in the physical properties of 30S subunit observed in the present study. In the presence of both IF1 and IF3 (Figure 5C), both ΔFwater and ΔDwater seem to be representing those in the presence of only IF1 (Figure 5A) and those in the presence of only IF3 (Figure 5B). Furthermore, we observed ΔFwater and ΔDwater values in response to the addition of higher concentrations (750 and 1000 nM) of fMet-tRNAfMet in the presence of IF3 (Supporting Information, Figure S2). The ΔFwater value increased and the ΔDwater value decreased with increasing the concentrations of fMet-tRNAfMet. These are reasonable, as the binding of IF2/ GTP−fMet-tRNAfMet results in the increase in ΔFwater and the decrease in ΔDwater due to clamping of 30S subunit as shown in Figure 3C and the subsequent structural changes of 30S subunit in the presence of IF3 results in the decrease in ΔFwater and the increase in ΔDwater as shown in Figure 5B. Therefore, the increase of only the concentrations of fMet-tRNAfMet could result in a predominance of the ΔFwater increase and the ΔDwater decrease. Thus, two structural changes in 30S complex (1) caused by the binding of IF2/GTP-fMet-tRNAfMet and then (2) depending on association of IF3 occur sequentially, triggered by the binding of fMet-tRNAfMet. In Situ Monitoring of Conformational Changes of 30S Initiation Complex for IF3 Binding. If the 30S conformational change responding to the binding of fMet-tRNAfMet is affected by the presence of IF3, the binding of IF3 also could 5413

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ACKNOWLEDGMENTS We are thankful to Prof. T. Ueda for providing ribosome and plasmid vectors of factors for cell-free translation initiation. We thank Mr. Sugahara for help with experiments. This work was supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan, Grant-in-Aid for Scientific Research.

cause 30S conformational change. When IF3 was injected to 30S subunit on mRNA as shown in Figure 6A, the ΔFwater decreased (the mass increased) at first due to the IF3 binding onto the 30S complex and then increased (the apparent mass decreased). On the contrary, the ΔDwater increased (the elasticity increased) at first and then decreased, corresponding to changes in 30S subunit caused by the binding of IF3. These results suggest that the IF3 binding could induce changes in physical property, dehydration, and increase in elasticity. These responses are in agreement with the reciprocal changes in the subsequent responses of 30S subunit in the presence of IF3, as in Figure 5B, suggesting that the responses could depend on association with IF3 to 30S subunit. In addition, we confirmed that the binding of fMet-tRNAfMet resulted in the increase in ΔFwater (dehydration) and the increase in ΔDwater (the viscosity increase), indicating that the two reactions associated with the binding of fMet-tRNAfMet and the release of IF3 occur simultaneously at complex time courses (Figure 6B). The results of the present study indicate that the binding of IF3 causes dehydration and increase in elasticity of 30S subunit bound on mRNA. The hydration and viscoelasticity of 30S subunit were also influenced by the presence of IF1. The addition of fMet-tRNAfMet to 30S subunit complex together with IF2/GTP resulted in other changes in the physical property (the dehydration and the elasticity increase) and caused two sequential changes on the time course in the presence of IF3, suggesting that the various states in the physical properties of viscoelastic 30S subunit is regulated by the binding of the fMet-tRNAfMet, IF1, IF2/GTP, and IF3.

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CONCLUSION We employed the energy dissipation measurement using QCMadmittance (QCM-A) for in situ monitoring of structural changes of pre-30S translation initiation complex in response to the binding of fMet-tRNAfMet in aqueous solution. The results obtained in this study indicate that the addition of fMettRNAfMet could cause changes in the physical property (the dehydration and the elasticity increase) of 30S subunit bound on the mRNA-immobilized QCM in the presence of IF2/GTP and/or IF3. These observations provide evidence that the conformational changes in 30S ribosomal subunit triggered by fMet-tRNAfMet operate as a switch to regulate in an orderly manner the process of construction of the translation initiation complex. A real-time monitoring of the conformational changes of a biomolecule in aqueous solution is helpful to understand the regulatory mechanism of biosystems. ASSOCIATED CONTENT

S Supporting Information *

Experimental results on a 30S subunit-immobilized QCM plate (Figure S1) and QCM data showing an effect of fMet-tRNAfMet concentration on conformational changes in 30S subunit (Figure S2). This material is available free of charge via the Internet at http://pubs.acs.org/.

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The authors declare no competing financial interest. 5414

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