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Structural studies of E. coli ribosomes by spectroscopic techniques: A specialized review
Spectrochimica Acta Part A 62 (2005) 1070–1080
Review
Structural studies of E. coli ribosomes by spectroscopic techniques: A specialized review Adalberto Bonicontro a , Gianfranco Risuleo a,b,∗ b
a INFM-CRS SOFT, Dipartimento di Fisica, Universit` a di Roma “La Sapienza”, P.le A. Moro 2, I-00185 Roma, Italy Dipartimento di Genetica e Biologia Molecolare, Universit`a di Roma “La Sapienza”, P.le A. Moro 2, I-00185 Roma, Italy
Received 5 April 2005; accepted 15 April 2005
Abstract We present a review on our interdisciplinary line of research based on strategies of molecular biology and biophysics. These have been applied to the study of the prokaryotic ribosome of the bacterium Escherichia coli. Our investigations on this organelle have continued for more than a decade and we have adopted different spectroscopic biophysical techniques such as: dielectric and fluorescence spectroscopy as well as light scattering (photon correlation spectroscopy). Here we report studies on the whole 70S ribosomes and on the separated subunits 30S and 50S. Our results evidence intrinsic structural features of the subunits: the small shows a more “floppy” structure, while the large one appears to be more rigid. Also, an inner “kernel” formed by the RNA/protein association is found within the ribosome. This kernel is surrounded by a ribonucleoprotein complex more exposed to the solvent. Initial analyses were done on the so called Kaldtschmit–Wittmann ribosome: more recently we have extended the studies to the “tight couple” ribosome known for its better functional performance in vitro. Data evidence a phenomenological correlation between the differential biological activity and the intrinsic structural properties of the two-ribosome species. Finally, investigations were also conducted on particles treated at sub-denaturing temperatures and on ribosomes partially deproteinized by salt treatment (ribosomal cores). Results suggest that the thermal treatment and the selective removal of proteins cause analogous structural alterations. © 2005 Elsevier B.V. All rights reserved. Keywords: Ribosome; Dielectric spectroscopy; Fluorescence; Light scattering
Contents 1.
2.
∗
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Ribosome purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. Dielectric spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3. Fluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4. Light scattering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5. Differential scanning calorimetry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Ribosome complexity and definition of an extremely stable internal structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Effect of the thermal treatment on ribosomes exposed at sub-denaturing temperatures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Comparison between thermally treated ribosomes and ribosomal cores . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. “Tight couple” and “loose couple” ribosomes: a structural and comparative study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. A concise overview of spectroscopic studies on ribosomes present in literature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Corresponding author. Tel.: +39 06 49912234; fax: +39 06 4440812. E-mail address: [email protected] (G. Risuleo).
1386-1425/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.saa.2005.04.032
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1. Introduction Ribosomes are organelles that play a fundamental role in protein biosynthesis: they are found in all living organisms. The activity of these particles is necessary for the formation of the peptide bond during the polymerization, in vitro and in vivo, of activated amino acid precursors into proteins. The ribosome of the mesophilic bacterium Escherichia coli is probably the most thoroughly investigated. Its structure is extremely complex since the particle is formed by more than 50 different proteins, also known as r-proteins and three RNA polymers (by analogy defined r-RNA). These three molecules have different sequences and molecular weights, in addition they present abundant double helical tracts. During protein synthesis a number of protein factors and ancillary small molecules are required for the completion of the peptide chain. A vast knowledge exists on this organelle [1–8]. Also, the three-dimensional organization was more ˚ resolution [9,10]. Furthermore, recently investigated at 5 A ˚ resolution was attained for the 50S subunit of the a 2.4 A extremophyle Haloarcula marismortui [11]: nevertheless the structure/function relationships have not been yet fully elucidated. This biological supra-molecular object has been studied by a variety of approaches and experimental strategies, including biochemistry, molecular biology and genetics, as well as chemico-physical ones (see for instance [3,5]). For longer than a decade we focused our study on the structural properties of the ribosome using a strategy based on biophysical spectroscopic techniques, i.e. dielectric and fluorescence spectroscopy as well as light scattering (photon correlation spectroscopy); these studies were also supported by differential scanning microcalorimetry. In this review we present the state of the art of our interdisciplinary line of research. For a better understanding of the results presented here, we proceed with a synthetic survey of the principal experimental techniques.
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Ribosomes with higher functional activity in vitro are obtained according to the method reported in [14]. These particles are defined tight couples (TC). Tight couple ribosomes and subunit particles were a generous gift of Knud Nierhaus (Max-Planck-Institut f¨ur Molekulare Genetik, Berlin). Cores were obtained according to a standard procedure based on the resuspension of the ribosomes in a solution of adequate LiCl concentration. Solutions were stirred at 4 ◦ C for at least 6 h, concentrated by high-speed centrifugation. Ribosomal pellets were resuspend and dialyzed exhaustively against measuring buffer (reported above). 1.2. Dielectric spectroscopy Dielectric spectroscopy [DS] was performed at radiofrequency ranges where biological systems normally exhibit characteristic relaxation processes. Persistence length of poly-electrolytes and structural protein properties, like hydrodynamic radius and electric dipole moment, can be for example measured. The measuring apparatus consists in a computer controlled Hewlett-Packard impedance analyzer Mod. 4194A used in the 0.1–100 MHz range. The measuring cell, previously described [15], is a section of a cylindrical wave-guide, which can be partially filled with the sample solution. The system behaves as wave guide excited far beyond its cut off frequency mode and therefore only the stray field of the coaxial line wave guide transition is used in the measurement. Cell constants were determined by an interpolation method based on measurements with electrolyte solutions of known conductivity similar to those of the samples under test, following a standard procedure [16,17]. The errors were within 1% both on the real and the imaginary part of the dielectric constant. The measuring cell was thermally controlled within 0.1 ◦ C. 1.3. Fluorescence
1.1. Ribosome purification Crude ribosomes were prepared as previously reported and subjected to two subsequent washings with 1 M NH4 Cl [12]. This procedure produces the “classical” Kaltschmidt–Wittmann ribosome also known as loose couple (LC). Prior to each dielectric measurement ribosomes were dialyzed against measuring buffer (0.8 mM MgCl2 , 3 mM KCl, 1 mM Tris–HCl pH 7.5). This buffer is required by the dielectric spectroscopy measurements that must be performed at as low ion strength as possible. Integrity of ribosomal particles in the measuring buffer was monitored also by sucrose density gradients [13]. Since resuspension in this buffer did not cause major structural rearrangements, it was used throughout the experimental measurements. In some experiments Mg2+ was substituted by a double concentration of Na+ thus producing a particle that we defined “relaxed”. Loose couple subunits were purified by zonal sucrose gradient centrifugation at low Mg2+ .
Measurements were done in different experimental conditions using a Perkin-Elmer LS50 luminescence spectrometer. The specific fluorophore ethidium bromide (EB) was added to suspensions of 30S and 50S separated subunits as well as 70S whole ribosomes. Static fluorescence measurements were performed in the presence of EB (excitation λ was 510 nm, emission was monitored at 600 nm). This drug intercalates mainly in the double stranded regions of rRNA. Variations of the intercalation are related to conformational changes of the particle. Also, the degree of exposure of nucleic acid to the solvent can be assessed by EB fluorescence [18–20]. 1.4. Light scattering Photon correlation spectroscopy was performed at a scattering angle of 90◦ , using a Brookhaven digital correlator set up BI9000AT equipped with 10 mW He–Ne laser (λ = 632.8 nm). Ribosome particle concentration (about
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1.8 mg/ml corresponding to 1013 particles/ml) was low enough to avoid particle/particle interactions and multiple scattering. For polydisperse systems, the scattered electric field autocorrelation function may be expressed as the Laplace transform of the decay rate distribution function G(). The relaxation times 1/ were found using the standard data analysis program CONTIN [21–23]. 1.5. Differential scanning calorimetry This technique allows a direct measurement of the energy change upon demolition of the particle with increasing temperature [24–27]. Experiments were performed by a differential scanning microcalorimeter 11 Setaram (Lyon, France) at a scan rate of 0.5 ◦ C/min, starting from 25 to 100 ◦ C. We shall report in Section 2, data obtained by this technique that corroborate the results of the spectroscopic measurements.
2. Results and discussion 2.1. Ribosome complexity and definition of an extremely stable internal structure The role of magnesium on the biological activity, structure and subunit association of ribosomes, is very well documented in “classical” literature [6,7]. We found that E. coli 70S ribosomes prepared according to the “classical” protocol of Kaltschmidt and Wittmann (LC) exhibit two distinctive dielectric relaxations, indicated by arrows (Fig. 1), the first in the kHz and the second in the MHz range [28]. The experimental data were fitted with a standard procedure based on a series of subsequent Cole–Cole relaxations [29]. The low frequency dispersion is strongly dependent on the presence of Mg2+ , while the relaxation time of the higher frequency process, is rather constant in all experimental conditions. As a matter of fact, when Mg2+ is eliminated from the buffer the low frequency relaxation is no longer monitored, while the one in the MHz range persists. The model originally proposed by Mandel, associates
Fig. 1. Permittivity ε vs. frequency of an E. coli 70S ribosome suspension. Frequency range 10 kHz–1 GHz. Temperature 25 ◦ C. Particle concentration was 5 mg/ml.
the dispersion phenomena in poly-electrolytes to counter-ion movements at the interface macromolecule/solvent: the polyion is represented as a sequence of thin rod like subunits of identical length b (“subunit b”) [30,31]. This parameter is assumed to be independent of the molecular weight for a macromolecule with a sufficiently high degree of polymerization. Counter-ions are free to move along the subunits, but cannot cross them unless a potential barrier, at the junction between each subunit, is bypassed. An external electric field perturbs the uniform distribution of counter-ions and consequently induces a dipole moment. We applied this model to ribosomal particles considering the counter-ion movements along traits of rRNA exposed to the solvent. If magnesium is substituted by sodium, the low frequency relaxation falls outside our measuring window: this can be rationalized as a higher exposure of rRNA to the solvent. The second relaxation is plausibly associated to the r-proteins interacting with rRNA. We corroborated these data by fluorescence studies evidencing that the absence of Mg2+ ions triggers a conformational alteration of the rRNA. The fluorescence intensity of the bound EB is definitely lower in the presence of magnesium and nearly independent of the concentration of rRNA, denoting a near zero association constant. The absence of magnesium produces, on the contrary, a higher fluorescence intensity and saturation is reached as a function of ribosome concentration (Fig. 2). In the case of the separated subunits, fluorescence is also minimal when Mg2+ is present. On the contrary without Mg2+ in the 30S and in the 50S, saturation is reached nearly at the same rRNA concentration and the same fluorescence intensity is produced. In conclusion in the
Fig. 2. Fluorescence intensity of ethidium bromide bound to ribosomes and ribosomal subunits as a function of rRNA concentration. () and (䊉) 70S particles. () and () 50S subunits. () and () 30S subunits. Full symbols: measurement in the presence of Mg2+ ; empty symbols without Mg2+ . The concentration of EB was 5 × 10−7 M.
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Fig. 3. Section A: Permittivity ε (upper panel) and dielectric loss ε (lower panel) vs. frequency of native 70S () and particles treated with RNase (). Section B: Permittivity ε (upper panel) and dielectric loss ε (lower panel) vs. frequency of native 70S () and core particles (). Particle concentration was 10 mg/ml. Measuring buffer: 0.8 mM MgCl2 , 1 mM Tris–HCl pH 7.5.
absence of this ion, the double helical traits of the ribosomal RNA are actually more extensively exposed to the solvent [18]. The whole 70S ribosome saturates at a much higher rRNA concentration and the intensity does not reach the same level as in the separated subunits. This indicates that, even in the absence of magnesium, the two subunits are still somehow interacting. The double stranded rRNA, which is essentially the portion of the molecule interacting with EB, is directly involved in the apparent integrity of the entire 70S particle. This result seems to contradict the common notion that lack of magnesium causes physical subunit separation. As a matter of fact we may observe a partition of the two subunits, lethal for the biological function, but that does not imply an authentic spatial separation. The behavior of ribosomal particles subjected to treatments that altered either the rRNA or the r-protein moiety, was also studied: these treatments cause controlled and limited demolition of the rRNA or selective loss of ribosomal proteins which gives rise to the so called “ribosomal core”. The results of DS show that the action of the nuclease (RNase), which hydrolizes the phophodiester bond, is exerted only on the low frequency dispersion. This dispersion is shifted towards higher frequencies with a reduction of dielectric increment (Fig. 3, Section A). In this case, unlike what previously reported for the untreated particles, the shift of relaxation frequency derives from a decreased RNA exposure to the solvent, as one would expect for a partially demolished RNA molecule. In the case of the cores, obtained treating the native ribosomes with increasing concentrations of LiCl, on the contrary, the first relaxation shifts to lower frequencies with a higher dielectric increment, which implies a significant increase of the exposed RNA [32]. The comparison between native ribosomes and cores shows that at low concentration of salt, essentially only the first low frequency relaxation is affected (Fig. 3, Section B). Table 1 summarizes the data of the
best fits referred to RNase treated ribosomes and ribosomal cores. The persistence of the second dispersion was ascribed to an intrinsic stability of a ribosomal “nucleus” that we defined: kernel. The treatment of ribosomes with increasing concentrations of LiCl has effect also on the second dispersion which is indicative of a partial loosening of the protein/RNA complex only above a critical concentration [23]. The treatment below this critical concentration removes most efficiently the following proteins from the small subunit: S1, S3, S9, S10, S20. From the large subunit, on the other hand, proteins L1, L8, L9 and L10 are eliminated. The role of these ribosomal proteins was addressed in a number of studies on the structure/function relationships in E. coli ribosome, showing that their elimination causes a severe loss of function [1,6–8,33,34]. The above-mentioned proteins, presumably, play little role for the maintenance of the “basic” ribosomal kernel, therefore this structure is maintained as in the integer particle. Differential Scanning Calorimetry [DSC] was also used as a support technique. Results obtained by DSC evidence two levels of structural organization [13]. A typical thermal scan of E. coli 70S ribosomes produces a highly structured pattern with two well-defined transitions occurring at different temperatures (Fig. 4A). The denaturation profile describes the thermal demolition of the RNA/protein complex, since proteins alone do not contribute significantly to calorimetric Table 1 Relaxation frequencies and dielectric increments of the two dispersions exhibited by native, RNase-treated samples and ribosomal cores (ribosome concentration was 10 mg/ml) Sample
f1∗ (kHz)
ε1
f2∗ (MHz)
ε2
Native 70S RNase treatment Ribosome cores
260 ± 20 490 ± 60 180 ± 20
19 ± 1 13 ± 3 29 ± 2
2.8 ± 0.6 3.1 ± 0.9 2.6 ± 0.7
10 ± 1 9±2 11 ± 2
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measurement at this particle concentration. The two transitions in whole ribosomes are irreversible and the two distinctive phenomena are thermally separated and independent. Calorimetric experiments were also performed on separated subunits. The large one behaves substantially as the whole 70S. This similarity suggests that within the entire ribosome and the 50S subunit, the spatial arrangement of rRNA and proteins is essentially the same. The thermal denaturation profile of the 30S subunit consists in a non-structured thermogram strongly resembling the free RNA in solution (Fig. 4B and C, respectively). An exothermic peak during the transition indicates aggregation phenomena that make impossible the analysis of the thermogram. A similar phenomenon is present also in the 50S, but it appears when the transition is
concluded. The thermal pattern exhibited by the 30S subunit suggests an intrinsic instability of the particle. The calorimetric data further support the idea that the 50S is more rigid while the 30S shows “floppier” characteristics. In conclusion, the two separated transitions are not ascribable to the each individual subunit since the peaks may be still observed in the 50S particle whereas the thermogram of the isolated 30S does not show any sharp and well-defined transition. The DSC approach was used also to investigate ribosomal particles treated with RNase or cores deprived of proteins as a consequence of LiCl treatment [35,36]. The persistence of the high temperature peak was observed in both cases (Fig. 5). The results are consistent with those obtained by DS and confirm the existence of the kernel. On the contrary, the first thermal transition tends to disappear. This implies that the partial demolition of rRNA or the extraction of surface proteins, possibly cause the disruption of weaker structural levels as also shown by DS data. 2.2. Effect of the thermal treatment on ribosomes exposed at sub-denaturing temperatures
Fig. 4. Thermal profile of excess heat capacity (Cpexc ) of 70S ribosomal particles (A); 50S (B) and 30S (C) ribosomal subunits. The curves were calculated by the subtraction of a baseline from the recorder tracing. Ribosome concentration was 5 mg/ml.
This investigation was carried out by dynamic light scattering in addition to fluorescence and dielectric spectroscopy [37]. Ribosomes were treated at a sub-denaturing temperature (70 ◦ C) for 2 and 4 min. This temperature was chosen on the basis of the previous calorimetric experiments that showed two thermal transitions occurring before the irreversible denaturation of the ribosome (above 70 ◦ C). Experimental data obtained by light scattering, show discrete populations of native ribosomes distributed as a function of the diffusion coefficient D (Fig. 6, lower panel). The presence of large ribosome aggregates and smaller particles, that could represent isolated subunits or ribosome fragments, are evident. Aggregates represent about 55% of the whole particle population and sub-ribosomal particles constitute 25%, the remaining 20% are authentic 70S ribosomes. This heterogeneity is not uncommon for the Kaltschmidt–Wittmann ribosomes (LC). After thermal treatment LC particles shift to lower values of diffusion coefficient, denoting strong phenomena of geometry alteration and possible processes of aggregation (Fig. 6, upper panel). The intensity of fluorescence emission of ethidium bromide (EB) was measured as a function of the fluorophore/ribosome molar ratio. The absence of a defined isosbestic point indicates two types of binding, that occur essentially with RNA, and that are of the van der Waals and/or electrostatic type. At high ion strength the second weak binding is abolished, which indicates its electrostatic nature: we assume that this binding is established with charges essentially located on r-proteins even though interactions with the phosphate backbone of the rRNA cannot be ruled out. The strong interaction occurs mainly with the double helical rRNA traits. In Fig. 7 the binding isotherms for native and thermally treated ribosomes are reported and Table 2 summarizes the results of the best fits based on two bindings. After
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Fig. 5. Thermal profile of excess heat capacity (Cpexc ). Native and RNase treated 70S: left lower and upper panels, respectively. Native and core particles: right lower and upper panels, respectively at same concentration as in the previous figure.
the thermal treatment, the overall number of binding sites increases and this recalls the phenomena already observed in DS measurements after removal of magnesium as well as after elimination of select proteins: therefore, the interpretation of a higher exposure of rRNA to the solvent can be considered
also in this case. As far as the dielectric measurements is concerning, data show that the treatment produces a decrease of the frequency of first dielectric relaxation. This implies an increase of the subunit length in the rRNA. In addition also the second relaxation is altered. This suggests a partial “loosening” of the protein/RNA complex as a consequence of the treatment at sub-denaturing temperatures (Fig. 8). 2.3. Comparison between thermally treated ribosomes and ribosomal cores The results presented above suggest that the thermal treatment at sub-denaturing temperatures induces significant
Fig. 6. Distribution of ribosomal particles as a function of the translational diffusion coefficient. Lower and upper panels show the native and thermally treated LC particles (4 min at 70 ◦ C). Ribosome concentration was 1.8 mg/ml corresponding to about 1013 particles/ml.
Fig. 7. Fluorescence measurements on LC particles. Binding isotherms: () native particles; () and () thermal treatment at 70 ◦ C for 2 and 4 min, respectively. The continuous lines are the result of a best fit based on the hypothesis of two different bindings fluorophore/ribosome. Ethidium bromide was at the fixed concentration 5 × 10−7 M. The concentration of ribosomes was varied up to a maximum of 0.1 mg/ml.
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Table 2 Number of sites excluded per mole of bound EthBr
70S LC Native 2 min treatment 4 min treatment
N1 (10−3 )
K1 (107 M−1 )
N2 (10−3 )
K2 (105 M−1 )
1.65 ± 0.05 2.50 ± 0.08 2.60 ± 0.10
1.00 ± 0.05 0.80 ± 0.10 0.80 ± 0.10
4.94 ± 0.08 3.34 ± 0.08 3.30 ± 0.20
5.00 ± 0.06 4.90 ± 0.10 5.00 ± 0.20
The association constants of the strong (N1 , K1 ) and weak (N2 , K2 ) binding for native and thermally treated (70 ◦ C) ribosomes are also shown. Data refer to LC ribosomes and their concentration is reported in the corresponding figure.
Fig. 8. Permittivity ε vs. frequency of 70S LC ribosomes. In the inset dielectric loss ε . () Native particles; () and () thermal treatment at 70 ◦ C for 2 and 4 min, respectively. Ribosome concentration was 10 mg/ml.
structural alterations on the ribosome particle that nevertheless maintains its average properties. The effect of these treatments was compared to the behavior of cores investigated by the three spectroscopic techniques illustrated above [23]. The core particle distribution as a function of D is reported in Fig. 9. It is worth noting that after treatment with LiCl the correspondence between diffusion coefficient (D) and
ribosome size, becomes critical because of possible conformational alterations promoted by the removal of proteins. However, two characteristic features are evident as compared to native particle distribution: the population of small particles (high D value) decreases while the one with low D value increases. This is most likely due to the formation of aggregates and, interestingly, similar phenomena were also observed after exposure to sub-denaturing temperatures [37]. Dielectric measurements conducted on native ribosomes and core particles show a decreasing trend of the first relaxation frequency whose consequences are amply discussed above. In the case of ribosomes treated with the highest LiCl concentration, the second dielectric relaxation is also affected. This denotes a partial destabilization of the internal ribosome structure (kernel). In conclusion, the consequences of thermal and high salt treatments show significant analogies consisting in a partial “loosening” of the protein/RNA complex. Fluorescence is also in support of these conclusions. In fact, the number of strong binding sites between fluorophore and rRNA increases significantly. The number of weak binding sites is reduced and this is ascribable to loss of surface proteins [23]. The results obtained with the three spectroscopic techniques discussed so far, fit very well together and indicate that the main target of the thermal treatment is represented by proteins. 2.4. “Tight couple” and “loose couple” ribosomes: a structural and comparative study
Fig. 9. Distribution of ribosomal particles as a function of the translational diffusion coefficient. Lower and upper panels show the native and 1.5 M LC core particles. Ribosome concentration was 1.8 mg/ml corresponding to about 1013 particles/ml.
In the studies presented so far, we used the “classical” Kaltschmidt–Wittmann protocol. This method has a very high yield and produces particles with a satisfactory functional activity. The so called “tight couples” (TC) are purified according to a different protocol and show a much higher biological activity in vitro. Therefore, we carried out a comparative structural study aimed at a phenomenological correlation between functional activity and intrinsic structural properties [38]. The TC 70S particles show a significant difference in the low frequency dispersion as compared to LC ribosomes (Fig. 10, the arrows point out the differences in the actual relaxation frequencies). In particular, the relaxation occurs at a higher frequency while the dielectric increment is lower. This implies that longer traits of rRNA are exposed in LC 70S and it suggests a decrease of particle compactness. Therefore,
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Fig. 10. Permittivity ε vs. frequency of 70S LC ribosomes (upper panel) and TC ribosomes (lower panel). The insets report dielectric loss ε . Arrows visualize the relaxation frequencies of the different samples. Particle concentration was 10 mg/ml.
Fig. 11. Permittivity ε vs. frequency of 30S LC subunits (upper panel) and 30S TC subunits (lower panel). The insets report dielectric loss ε . Arrows visualize the relaxation frequencies. Particle concentration was 10 mg/ml.
we conclude that TC are “denser” than LC particles. This conclusion, obtained by a very simple experimental strategy is in agreement with recent work obtained by solution X-ray and neutron scattering. In this work it was demonstrated that in the 30S the rRNA moiety is more anisometric than the subunit itself. Furthermore, the rRNA of the 50S subunit forms a compact core [39,40]. The same study was performed on the separate subunits obtained from both ribosome species. The 50S TC and LC show the same dielectric behavior and 30S TC resembles 70S since it produces two different dispersions. On the other hand, LC 30S subunits show only the second relaxation but shifted to a lower frequency (Fig. 11). As mentioned earlier, the first dispersion is very likely located out of our measuring window of frequencies [15]. These data are consistent with a “floppier” nature of the LC small ribosomal subunit as compared the homologous TC subunit. In conclusion, the structural differences monitored in the whole ribosome reside mainly in the small subunit. Microcalorimetric measurements on 70S and 50S obtained by the two different protocols, do not exhibit substantial differences in the thermal profile while, as already discussed, the small LC subunit produces an unstructured thermal profile. Particles from TC preparations exhibit, on the contrary, a well-defined peak that indicates a single denaturation event. In addition, the first denaturation peak in LC 70S occurs at a slightly lower temperature; this shows that the intrinsic floppiness of LC 30S affects the overall structure of the ribosome, in agreement with the dielectric data. Functional and structural transitions from TC to LC and vice versa were reported even though no important structural differences were evidenced [41]. An overall conclusion can
be drawn from these data: in TC particles the rRNA is less exposed than in LC particles; furthermore TC ribosomes have a more compact structure. Therefore, the loss of compactness observed in LC ribosomes may be related to their decrease of function in vitro. We investigated the configuration of both ribosome species prior to complete particle collapse. The strategy of these experiments is analogous to the one described previously on LC ribosomes. Also in this case the combination of the three biophysical approaches was adopted. Dynamic light scattering data show that native TC particles are found over a relatively narrow range of diffusion coefficients. About 30% of the whole population, accounts for 70S ribosomes: we remind that in the case of LC ribosomes 20% constituted the monomeric particle species. After the thermal treatment TCs’ form two major groups of particles. The first has a very low value of diffusion coefficient (D) and thus represents aggregates. The second one remains in a range of D that characterizes the native TC 70S and represents about 15% of the total (Fig. 12). On the contrary, as reported above, the thermal treatment of LC couples did not evidence a discrete particle population. A quantitative estimation in this case is impossible, since the thermal treatment produces significant alterations and therefore a form factor should be taken into account. Results of light scattering and dielectric spectroscopy measurements are totally consistent. The higher exposure of rRNA traits after thermal treatment is demonstrated by the increase of the subunit length b which is more relevant for LC than TC particles. The persistence of the second dielectric dispersion suggests a particular stability of the kernel in these latter particles (Figs. 8 and 13 and Table 3
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Fig. 12. Distribution of ribosomal particles as a function of the translational diffusion coefficient. Lower and upper panels show the native and thermally treated TC particles (4 min at 70 ◦ C). Ribosome concentration was 1.8 mg/ml corresponding to about 1013 particles/ml.
which summarizes all data concerning LC and TC 70S particles). Fluorescence measurements show that the second weak binding of EB is essentially the same for both thermally treated and untreated ribosome as far as TC particles is concerning. The number of strong binding sites increases after the treatment but this phenomenon is much less relevant for TC than for LC ribosomes (Figs. 7 and 14 and Tables 2 and 4 which summarize the data on LC and TC ribosomes). This further validates the notion that loose couples are more sensitive to the thermal treatment. The data conclusively demonstrate that the higher functional in vitro activity of TC ribosomes can be associated to intrinsic structural differences that imply a more resistant kernel and a reduced exposure of RNA to the solvent. The ribosomal machinery is obviously very complex and its fundamental role in protein synthesis is well established. Ribosomes are highly conserved throughout evolution and contain large amounts of RNA: more that 50% of the whole mass. Ribosomal RNA is directly involved in translation and its ribozyme role has been suggested. Also ribosomal RNA may be a molecular fossil dating back to the age of early molecular evolution when the RNA had a genetic and catalytic function [42–45]. The data discussed in this work seem to support this idea. However, it should be taken into account that the ribozyme role is still a matter of discussion [46,47]. 2.5. A concise overview of spectroscopic studies on ribosomes present in literature
Fig. 13. Permittivity ε vs. frequency of 70S TC ribosomes. In the inset dielectric loss ε . () Native particles; () and () thermal treatment at 70 ◦ C for 2 and 4 min, respectively. Particle concentration was 10 mg/ml.
A very large number of investigations on ribosomes by spectroscopic approaches, encompassing a great variety of experimental technologies, are present in literature. These studies were conducted on biological macromolecules and also on supramolecular structures as complex as ribosomes. It is therefore very difficult to take into account the abundant literature and to give a general panorama of the results. For instance, in their pioneering works Beychok and Tinoco [48,49] used optical rotatory dispersion and circular
Table 3 Dielectric parameters of the two dispersions observed in native and thermally treated (70 ◦ C) LC and TC particles ε1
ε2
f2 (106 Hz)
b (nm)
70S LC Native 8 ± 1 5.1 ± 0.4 2 min treatment 11 ± 1 4.2 ± 0.4 4 min treatment 19 ± 2 2.5 ± 0.3
13 ± 2 16 ± 2 26 ± 3
3.4 ± 0.6 2.8 ± 0.5 1.3 ± 0.3
73 ± 2 80 ± 3 104 ± 5
70S TC Native 5 ± 2 7.1 ± 0.6 2 min treatment 5 ± 2 7.2 ± 0.7 4 min treatment 15 ± 2 5.8 ± 0.3
16 ± 2 18 ± 3 19 ± 4
2.2 ± 0.6 2.0 ± 0.6 1.8 ± 0.6
62 ± 2 62 ± 3 69 ± 2
f1 (105 Hz)
The last column reports the estimate of the subunit length b. Ribosome concentration was 10 mg/ml.
Fig. 14. Fluorescence measurements on TC particles. Binding isotherms: () native particles; () and () thermal treatment at 70 ◦ C for 2 and 4 min, respectively. The continuous lines are the result of a best fit based on the hypothesis of two different bindings fluorophore/ribosome. Ethidium bromide was at the fixed concentration 5 × 10−7 M. The concentration of ribosomes was varied up to a maximum of 0.1 mg/ml.
A. Bonicontro, G. Risuleo / Spectrochimica Acta Part A 62 (2005) 1070–1080
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Table 4 Number of sites excluded per mole of bound EthBr
70S TC Native 2 min treatment 4 min treatment
N1 (10−3 )
K1 (107 M−1 )
N2 (10−3 )
K2 (105 M−1 )
1.59 ± 0.05 1.85 ± 0.09 2.20 ± 0.10
1.00 ± 0.05 0.80 ± 0.09 0.80 ± 0.10
5.48 ± 0.06 4.20 ± 0.08 3.10 ± 0.20
5.00 ± 0.07 5.00 ± 0.10 5.00 ± 0.20
The association constants of the strong (N1 , K1 ) and weak (N2 , K2 ) binding for native and thermally treated ribosomes are also shown. Data refer to TC ribosomes.
dichroism to investigate the structure of biopolymers as early as 1968 and 1970; also small angle X-ray dispersion, that permitted the elucidation of DNA structure, was applied to the study of solutions of biological supra-molecular objects [50]. Fluctuation spectroscopy, a more exotic investigation tool, found also a role in molecular biology [51]. We shall cite here some relevant and significant reviews reporting data that we consider more directly related to our studies. This summary, of course, will not be exhaustive. The seminal review “Components of bacterial ribosomes” by the late Prof. Heinz Gunther Wittmann can be a starting point of a review on the structure of the bacterial ribosome and the macromolecules that contribute to its function and structure [52]. Fluorescence measurements were also used in the investigation of the ribosomal structure and function: in particular, five different approaches were adopted, i.e.: emission intensity; emission wavelength maximum; fluorescence anisotropy; collisional quenching; and energy transfer. The results of these investigations allowed a description of the movement and conformation of the main biopolymers (tRNA, nascent peptide, and mRNA) involved in the formation of the peptide bond and in the polypeptide elongation [53]. Furthermore, Xray diffraction, NMR spectroscopy and electron microscopy gave an insight on the complex structure of the rRNA: for instance, ribosomal proteins L11 and S15 show that RNA/protein interactions are involved in the establishment of the ribosome structure at the junctions between ribosomal RNA helices [54–56]. The exchange-transferred nuclear Overhauser effect of NMR spectroscopy may be informative of the interaction between small ligand molecules and high-molecular-weight proteins or nucleic acids. Further applications of the method involve systems of great complexity, such as ribosomes [57]. Electron cryo-microscopy, although strictly speaking cannot be considered a spectroscopic technique, has assumed a growing importance in recent years. Many molecular structures of both prokaryotic and eukaryotic ribosomal complexes have been elucidated by this technique. In the case of E. coli 70S particle a resolution ˚ was reached, while in the case of the extremophyle of 11.5 A ˚ Thermus thermophylus the resolution was even higher (5.5 A) [42]. This allows the identification of structures as minute as RNA helices, peripheral proteins, and intersubunit bridges. Furthermore, analysis of the isolated ribosomal large subunit ˚ However, this 50S, was performed at a resolution of 7.5 A. is yet not sufficient to visualize the amino acid side chains [58,59]. Very recent developments of mass spectrometry
allow the study of protein nucleic acids interactions: in particular the stoichiometry of protein/nucleic acid interactions can be evaluated. This approach can be applied to complex macromolecular machines such as ribosomes [60]. A very high resolution was obtained by X-ray crystallography on ribosomes from mesophilic bacteria and from extremophiles; these works described the structure of the ribosome in various functional states [61–68]. A final consideration is that all the experimental investigations discussed so far, often are highly sophisticated and the preparation of the sample follows complex and costly protocols. It is therefore surprising that using our standard techniques, that complement perfectly each other, allowed us to do interesting observations on the structural properties of the ribosome and their phenomenological correlation to ribosome function.
Acknowledgements The authors are indebted to Cesare Cametti, Knud H. Nierhaus, Giuseppe Onori and Maria Grazia Ortore for their constant interest and active collaboration in the development of this research. The subjects covered in this review were included in the master degrees of several students whose valuable cooperation is also acknowledged.
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Review
Structural studies of E. coli ribosomes by spectroscopic techniques: A specialized review Adalberto Bonicontro a , Gianfranco Risuleo a,b,∗ b
a INFM-CRS SOFT, Dipartimento di Fisica, Universit` a di Roma “La Sapienza”, P.le A. Moro 2, I-00185 Roma, Italy Dipartimento di Genetica e Biologia Molecolare, Universit`a di Roma “La Sapienza”, P.le A. Moro 2, I-00185 Roma, Italy
Received 5 April 2005; accepted 15 April 2005
Abstract We present a review on our interdisciplinary line of research based on strategies of molecular biology and biophysics. These have been applied to the study of the prokaryotic ribosome of the bacterium Escherichia coli. Our investigations on this organelle have continued for more than a decade and we have adopted different spectroscopic biophysical techniques such as: dielectric and fluorescence spectroscopy as well as light scattering (photon correlation spectroscopy). Here we report studies on the whole 70S ribosomes and on the separated subunits 30S and 50S. Our results evidence intrinsic structural features of the subunits: the small shows a more “floppy” structure, while the large one appears to be more rigid. Also, an inner “kernel” formed by the RNA/protein association is found within the ribosome. This kernel is surrounded by a ribonucleoprotein complex more exposed to the solvent. Initial analyses were done on the so called Kaldtschmit–Wittmann ribosome: more recently we have extended the studies to the “tight couple” ribosome known for its better functional performance in vitro. Data evidence a phenomenological correlation between the differential biological activity and the intrinsic structural properties of the two-ribosome species. Finally, investigations were also conducted on particles treated at sub-denaturing temperatures and on ribosomes partially deproteinized by salt treatment (ribosomal cores). Results suggest that the thermal treatment and the selective removal of proteins cause analogous structural alterations. © 2005 Elsevier B.V. All rights reserved. Keywords: Ribosome; Dielectric spectroscopy; Fluorescence; Light scattering
Contents 1.
2.
∗
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Ribosome purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. Dielectric spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3. Fluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4. Light scattering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5. Differential scanning calorimetry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Ribosome complexity and definition of an extremely stable internal structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Effect of the thermal treatment on ribosomes exposed at sub-denaturing temperatures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Comparison between thermally treated ribosomes and ribosomal cores . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. “Tight couple” and “loose couple” ribosomes: a structural and comparative study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. A concise overview of spectroscopic studies on ribosomes present in literature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Corresponding author. Tel.: +39 06 49912234; fax: +39 06 4440812. E-mail address: [email protected] (G. Risuleo).
1386-1425/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.saa.2005.04.032
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1. Introduction Ribosomes are organelles that play a fundamental role in protein biosynthesis: they are found in all living organisms. The activity of these particles is necessary for the formation of the peptide bond during the polymerization, in vitro and in vivo, of activated amino acid precursors into proteins. The ribosome of the mesophilic bacterium Escherichia coli is probably the most thoroughly investigated. Its structure is extremely complex since the particle is formed by more than 50 different proteins, also known as r-proteins and three RNA polymers (by analogy defined r-RNA). These three molecules have different sequences and molecular weights, in addition they present abundant double helical tracts. During protein synthesis a number of protein factors and ancillary small molecules are required for the completion of the peptide chain. A vast knowledge exists on this organelle [1–8]. Also, the three-dimensional organization was more ˚ resolution [9,10]. Furthermore, recently investigated at 5 A ˚ resolution was attained for the 50S subunit of the a 2.4 A extremophyle Haloarcula marismortui [11]: nevertheless the structure/function relationships have not been yet fully elucidated. This biological supra-molecular object has been studied by a variety of approaches and experimental strategies, including biochemistry, molecular biology and genetics, as well as chemico-physical ones (see for instance [3,5]). For longer than a decade we focused our study on the structural properties of the ribosome using a strategy based on biophysical spectroscopic techniques, i.e. dielectric and fluorescence spectroscopy as well as light scattering (photon correlation spectroscopy); these studies were also supported by differential scanning microcalorimetry. In this review we present the state of the art of our interdisciplinary line of research. For a better understanding of the results presented here, we proceed with a synthetic survey of the principal experimental techniques.
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Ribosomes with higher functional activity in vitro are obtained according to the method reported in [14]. These particles are defined tight couples (TC). Tight couple ribosomes and subunit particles were a generous gift of Knud Nierhaus (Max-Planck-Institut f¨ur Molekulare Genetik, Berlin). Cores were obtained according to a standard procedure based on the resuspension of the ribosomes in a solution of adequate LiCl concentration. Solutions were stirred at 4 ◦ C for at least 6 h, concentrated by high-speed centrifugation. Ribosomal pellets were resuspend and dialyzed exhaustively against measuring buffer (reported above). 1.2. Dielectric spectroscopy Dielectric spectroscopy [DS] was performed at radiofrequency ranges where biological systems normally exhibit characteristic relaxation processes. Persistence length of poly-electrolytes and structural protein properties, like hydrodynamic radius and electric dipole moment, can be for example measured. The measuring apparatus consists in a computer controlled Hewlett-Packard impedance analyzer Mod. 4194A used in the 0.1–100 MHz range. The measuring cell, previously described [15], is a section of a cylindrical wave-guide, which can be partially filled with the sample solution. The system behaves as wave guide excited far beyond its cut off frequency mode and therefore only the stray field of the coaxial line wave guide transition is used in the measurement. Cell constants were determined by an interpolation method based on measurements with electrolyte solutions of known conductivity similar to those of the samples under test, following a standard procedure [16,17]. The errors were within 1% both on the real and the imaginary part of the dielectric constant. The measuring cell was thermally controlled within 0.1 ◦ C. 1.3. Fluorescence
1.1. Ribosome purification Crude ribosomes were prepared as previously reported and subjected to two subsequent washings with 1 M NH4 Cl [12]. This procedure produces the “classical” Kaltschmidt–Wittmann ribosome also known as loose couple (LC). Prior to each dielectric measurement ribosomes were dialyzed against measuring buffer (0.8 mM MgCl2 , 3 mM KCl, 1 mM Tris–HCl pH 7.5). This buffer is required by the dielectric spectroscopy measurements that must be performed at as low ion strength as possible. Integrity of ribosomal particles in the measuring buffer was monitored also by sucrose density gradients [13]. Since resuspension in this buffer did not cause major structural rearrangements, it was used throughout the experimental measurements. In some experiments Mg2+ was substituted by a double concentration of Na+ thus producing a particle that we defined “relaxed”. Loose couple subunits were purified by zonal sucrose gradient centrifugation at low Mg2+ .
Measurements were done in different experimental conditions using a Perkin-Elmer LS50 luminescence spectrometer. The specific fluorophore ethidium bromide (EB) was added to suspensions of 30S and 50S separated subunits as well as 70S whole ribosomes. Static fluorescence measurements were performed in the presence of EB (excitation λ was 510 nm, emission was monitored at 600 nm). This drug intercalates mainly in the double stranded regions of rRNA. Variations of the intercalation are related to conformational changes of the particle. Also, the degree of exposure of nucleic acid to the solvent can be assessed by EB fluorescence [18–20]. 1.4. Light scattering Photon correlation spectroscopy was performed at a scattering angle of 90◦ , using a Brookhaven digital correlator set up BI9000AT equipped with 10 mW He–Ne laser (λ = 632.8 nm). Ribosome particle concentration (about
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1.8 mg/ml corresponding to 1013 particles/ml) was low enough to avoid particle/particle interactions and multiple scattering. For polydisperse systems, the scattered electric field autocorrelation function may be expressed as the Laplace transform of the decay rate distribution function G(). The relaxation times 1/ were found using the standard data analysis program CONTIN [21–23]. 1.5. Differential scanning calorimetry This technique allows a direct measurement of the energy change upon demolition of the particle with increasing temperature [24–27]. Experiments were performed by a differential scanning microcalorimeter 11 Setaram (Lyon, France) at a scan rate of 0.5 ◦ C/min, starting from 25 to 100 ◦ C. We shall report in Section 2, data obtained by this technique that corroborate the results of the spectroscopic measurements.
2. Results and discussion 2.1. Ribosome complexity and definition of an extremely stable internal structure The role of magnesium on the biological activity, structure and subunit association of ribosomes, is very well documented in “classical” literature [6,7]. We found that E. coli 70S ribosomes prepared according to the “classical” protocol of Kaltschmidt and Wittmann (LC) exhibit two distinctive dielectric relaxations, indicated by arrows (Fig. 1), the first in the kHz and the second in the MHz range [28]. The experimental data were fitted with a standard procedure based on a series of subsequent Cole–Cole relaxations [29]. The low frequency dispersion is strongly dependent on the presence of Mg2+ , while the relaxation time of the higher frequency process, is rather constant in all experimental conditions. As a matter of fact, when Mg2+ is eliminated from the buffer the low frequency relaxation is no longer monitored, while the one in the MHz range persists. The model originally proposed by Mandel, associates
Fig. 1. Permittivity ε vs. frequency of an E. coli 70S ribosome suspension. Frequency range 10 kHz–1 GHz. Temperature 25 ◦ C. Particle concentration was 5 mg/ml.
the dispersion phenomena in poly-electrolytes to counter-ion movements at the interface macromolecule/solvent: the polyion is represented as a sequence of thin rod like subunits of identical length b (“subunit b”) [30,31]. This parameter is assumed to be independent of the molecular weight for a macromolecule with a sufficiently high degree of polymerization. Counter-ions are free to move along the subunits, but cannot cross them unless a potential barrier, at the junction between each subunit, is bypassed. An external electric field perturbs the uniform distribution of counter-ions and consequently induces a dipole moment. We applied this model to ribosomal particles considering the counter-ion movements along traits of rRNA exposed to the solvent. If magnesium is substituted by sodium, the low frequency relaxation falls outside our measuring window: this can be rationalized as a higher exposure of rRNA to the solvent. The second relaxation is plausibly associated to the r-proteins interacting with rRNA. We corroborated these data by fluorescence studies evidencing that the absence of Mg2+ ions triggers a conformational alteration of the rRNA. The fluorescence intensity of the bound EB is definitely lower in the presence of magnesium and nearly independent of the concentration of rRNA, denoting a near zero association constant. The absence of magnesium produces, on the contrary, a higher fluorescence intensity and saturation is reached as a function of ribosome concentration (Fig. 2). In the case of the separated subunits, fluorescence is also minimal when Mg2+ is present. On the contrary without Mg2+ in the 30S and in the 50S, saturation is reached nearly at the same rRNA concentration and the same fluorescence intensity is produced. In conclusion in the
Fig. 2. Fluorescence intensity of ethidium bromide bound to ribosomes and ribosomal subunits as a function of rRNA concentration. () and (䊉) 70S particles. () and () 50S subunits. () and () 30S subunits. Full symbols: measurement in the presence of Mg2+ ; empty symbols without Mg2+ . The concentration of EB was 5 × 10−7 M.
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Fig. 3. Section A: Permittivity ε (upper panel) and dielectric loss ε (lower panel) vs. frequency of native 70S () and particles treated with RNase (). Section B: Permittivity ε (upper panel) and dielectric loss ε (lower panel) vs. frequency of native 70S () and core particles (). Particle concentration was 10 mg/ml. Measuring buffer: 0.8 mM MgCl2 , 1 mM Tris–HCl pH 7.5.
absence of this ion, the double helical traits of the ribosomal RNA are actually more extensively exposed to the solvent [18]. The whole 70S ribosome saturates at a much higher rRNA concentration and the intensity does not reach the same level as in the separated subunits. This indicates that, even in the absence of magnesium, the two subunits are still somehow interacting. The double stranded rRNA, which is essentially the portion of the molecule interacting with EB, is directly involved in the apparent integrity of the entire 70S particle. This result seems to contradict the common notion that lack of magnesium causes physical subunit separation. As a matter of fact we may observe a partition of the two subunits, lethal for the biological function, but that does not imply an authentic spatial separation. The behavior of ribosomal particles subjected to treatments that altered either the rRNA or the r-protein moiety, was also studied: these treatments cause controlled and limited demolition of the rRNA or selective loss of ribosomal proteins which gives rise to the so called “ribosomal core”. The results of DS show that the action of the nuclease (RNase), which hydrolizes the phophodiester bond, is exerted only on the low frequency dispersion. This dispersion is shifted towards higher frequencies with a reduction of dielectric increment (Fig. 3, Section A). In this case, unlike what previously reported for the untreated particles, the shift of relaxation frequency derives from a decreased RNA exposure to the solvent, as one would expect for a partially demolished RNA molecule. In the case of the cores, obtained treating the native ribosomes with increasing concentrations of LiCl, on the contrary, the first relaxation shifts to lower frequencies with a higher dielectric increment, which implies a significant increase of the exposed RNA [32]. The comparison between native ribosomes and cores shows that at low concentration of salt, essentially only the first low frequency relaxation is affected (Fig. 3, Section B). Table 1 summarizes the data of the
best fits referred to RNase treated ribosomes and ribosomal cores. The persistence of the second dispersion was ascribed to an intrinsic stability of a ribosomal “nucleus” that we defined: kernel. The treatment of ribosomes with increasing concentrations of LiCl has effect also on the second dispersion which is indicative of a partial loosening of the protein/RNA complex only above a critical concentration [23]. The treatment below this critical concentration removes most efficiently the following proteins from the small subunit: S1, S3, S9, S10, S20. From the large subunit, on the other hand, proteins L1, L8, L9 and L10 are eliminated. The role of these ribosomal proteins was addressed in a number of studies on the structure/function relationships in E. coli ribosome, showing that their elimination causes a severe loss of function [1,6–8,33,34]. The above-mentioned proteins, presumably, play little role for the maintenance of the “basic” ribosomal kernel, therefore this structure is maintained as in the integer particle. Differential Scanning Calorimetry [DSC] was also used as a support technique. Results obtained by DSC evidence two levels of structural organization [13]. A typical thermal scan of E. coli 70S ribosomes produces a highly structured pattern with two well-defined transitions occurring at different temperatures (Fig. 4A). The denaturation profile describes the thermal demolition of the RNA/protein complex, since proteins alone do not contribute significantly to calorimetric Table 1 Relaxation frequencies and dielectric increments of the two dispersions exhibited by native, RNase-treated samples and ribosomal cores (ribosome concentration was 10 mg/ml) Sample
f1∗ (kHz)
ε1
f2∗ (MHz)
ε2
Native 70S RNase treatment Ribosome cores
260 ± 20 490 ± 60 180 ± 20
19 ± 1 13 ± 3 29 ± 2
2.8 ± 0.6 3.1 ± 0.9 2.6 ± 0.7
10 ± 1 9±2 11 ± 2
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measurement at this particle concentration. The two transitions in whole ribosomes are irreversible and the two distinctive phenomena are thermally separated and independent. Calorimetric experiments were also performed on separated subunits. The large one behaves substantially as the whole 70S. This similarity suggests that within the entire ribosome and the 50S subunit, the spatial arrangement of rRNA and proteins is essentially the same. The thermal denaturation profile of the 30S subunit consists in a non-structured thermogram strongly resembling the free RNA in solution (Fig. 4B and C, respectively). An exothermic peak during the transition indicates aggregation phenomena that make impossible the analysis of the thermogram. A similar phenomenon is present also in the 50S, but it appears when the transition is
concluded. The thermal pattern exhibited by the 30S subunit suggests an intrinsic instability of the particle. The calorimetric data further support the idea that the 50S is more rigid while the 30S shows “floppier” characteristics. In conclusion, the two separated transitions are not ascribable to the each individual subunit since the peaks may be still observed in the 50S particle whereas the thermogram of the isolated 30S does not show any sharp and well-defined transition. The DSC approach was used also to investigate ribosomal particles treated with RNase or cores deprived of proteins as a consequence of LiCl treatment [35,36]. The persistence of the high temperature peak was observed in both cases (Fig. 5). The results are consistent with those obtained by DS and confirm the existence of the kernel. On the contrary, the first thermal transition tends to disappear. This implies that the partial demolition of rRNA or the extraction of surface proteins, possibly cause the disruption of weaker structural levels as also shown by DS data. 2.2. Effect of the thermal treatment on ribosomes exposed at sub-denaturing temperatures
Fig. 4. Thermal profile of excess heat capacity (Cpexc ) of 70S ribosomal particles (A); 50S (B) and 30S (C) ribosomal subunits. The curves were calculated by the subtraction of a baseline from the recorder tracing. Ribosome concentration was 5 mg/ml.
This investigation was carried out by dynamic light scattering in addition to fluorescence and dielectric spectroscopy [37]. Ribosomes were treated at a sub-denaturing temperature (70 ◦ C) for 2 and 4 min. This temperature was chosen on the basis of the previous calorimetric experiments that showed two thermal transitions occurring before the irreversible denaturation of the ribosome (above 70 ◦ C). Experimental data obtained by light scattering, show discrete populations of native ribosomes distributed as a function of the diffusion coefficient D (Fig. 6, lower panel). The presence of large ribosome aggregates and smaller particles, that could represent isolated subunits or ribosome fragments, are evident. Aggregates represent about 55% of the whole particle population and sub-ribosomal particles constitute 25%, the remaining 20% are authentic 70S ribosomes. This heterogeneity is not uncommon for the Kaltschmidt–Wittmann ribosomes (LC). After thermal treatment LC particles shift to lower values of diffusion coefficient, denoting strong phenomena of geometry alteration and possible processes of aggregation (Fig. 6, upper panel). The intensity of fluorescence emission of ethidium bromide (EB) was measured as a function of the fluorophore/ribosome molar ratio. The absence of a defined isosbestic point indicates two types of binding, that occur essentially with RNA, and that are of the van der Waals and/or electrostatic type. At high ion strength the second weak binding is abolished, which indicates its electrostatic nature: we assume that this binding is established with charges essentially located on r-proteins even though interactions with the phosphate backbone of the rRNA cannot be ruled out. The strong interaction occurs mainly with the double helical rRNA traits. In Fig. 7 the binding isotherms for native and thermally treated ribosomes are reported and Table 2 summarizes the results of the best fits based on two bindings. After
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Fig. 5. Thermal profile of excess heat capacity (Cpexc ). Native and RNase treated 70S: left lower and upper panels, respectively. Native and core particles: right lower and upper panels, respectively at same concentration as in the previous figure.
the thermal treatment, the overall number of binding sites increases and this recalls the phenomena already observed in DS measurements after removal of magnesium as well as after elimination of select proteins: therefore, the interpretation of a higher exposure of rRNA to the solvent can be considered
also in this case. As far as the dielectric measurements is concerning, data show that the treatment produces a decrease of the frequency of first dielectric relaxation. This implies an increase of the subunit length in the rRNA. In addition also the second relaxation is altered. This suggests a partial “loosening” of the protein/RNA complex as a consequence of the treatment at sub-denaturing temperatures (Fig. 8). 2.3. Comparison between thermally treated ribosomes and ribosomal cores The results presented above suggest that the thermal treatment at sub-denaturing temperatures induces significant
Fig. 6. Distribution of ribosomal particles as a function of the translational diffusion coefficient. Lower and upper panels show the native and thermally treated LC particles (4 min at 70 ◦ C). Ribosome concentration was 1.8 mg/ml corresponding to about 1013 particles/ml.
Fig. 7. Fluorescence measurements on LC particles. Binding isotherms: () native particles; () and () thermal treatment at 70 ◦ C for 2 and 4 min, respectively. The continuous lines are the result of a best fit based on the hypothesis of two different bindings fluorophore/ribosome. Ethidium bromide was at the fixed concentration 5 × 10−7 M. The concentration of ribosomes was varied up to a maximum of 0.1 mg/ml.
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Table 2 Number of sites excluded per mole of bound EthBr
70S LC Native 2 min treatment 4 min treatment
N1 (10−3 )
K1 (107 M−1 )
N2 (10−3 )
K2 (105 M−1 )
1.65 ± 0.05 2.50 ± 0.08 2.60 ± 0.10
1.00 ± 0.05 0.80 ± 0.10 0.80 ± 0.10
4.94 ± 0.08 3.34 ± 0.08 3.30 ± 0.20
5.00 ± 0.06 4.90 ± 0.10 5.00 ± 0.20
The association constants of the strong (N1 , K1 ) and weak (N2 , K2 ) binding for native and thermally treated (70 ◦ C) ribosomes are also shown. Data refer to LC ribosomes and their concentration is reported in the corresponding figure.
Fig. 8. Permittivity ε vs. frequency of 70S LC ribosomes. In the inset dielectric loss ε . () Native particles; () and () thermal treatment at 70 ◦ C for 2 and 4 min, respectively. Ribosome concentration was 10 mg/ml.
structural alterations on the ribosome particle that nevertheless maintains its average properties. The effect of these treatments was compared to the behavior of cores investigated by the three spectroscopic techniques illustrated above [23]. The core particle distribution as a function of D is reported in Fig. 9. It is worth noting that after treatment with LiCl the correspondence between diffusion coefficient (D) and
ribosome size, becomes critical because of possible conformational alterations promoted by the removal of proteins. However, two characteristic features are evident as compared to native particle distribution: the population of small particles (high D value) decreases while the one with low D value increases. This is most likely due to the formation of aggregates and, interestingly, similar phenomena were also observed after exposure to sub-denaturing temperatures [37]. Dielectric measurements conducted on native ribosomes and core particles show a decreasing trend of the first relaxation frequency whose consequences are amply discussed above. In the case of ribosomes treated with the highest LiCl concentration, the second dielectric relaxation is also affected. This denotes a partial destabilization of the internal ribosome structure (kernel). In conclusion, the consequences of thermal and high salt treatments show significant analogies consisting in a partial “loosening” of the protein/RNA complex. Fluorescence is also in support of these conclusions. In fact, the number of strong binding sites between fluorophore and rRNA increases significantly. The number of weak binding sites is reduced and this is ascribable to loss of surface proteins [23]. The results obtained with the three spectroscopic techniques discussed so far, fit very well together and indicate that the main target of the thermal treatment is represented by proteins. 2.4. “Tight couple” and “loose couple” ribosomes: a structural and comparative study
Fig. 9. Distribution of ribosomal particles as a function of the translational diffusion coefficient. Lower and upper panels show the native and 1.5 M LC core particles. Ribosome concentration was 1.8 mg/ml corresponding to about 1013 particles/ml.
In the studies presented so far, we used the “classical” Kaltschmidt–Wittmann protocol. This method has a very high yield and produces particles with a satisfactory functional activity. The so called “tight couples” (TC) are purified according to a different protocol and show a much higher biological activity in vitro. Therefore, we carried out a comparative structural study aimed at a phenomenological correlation between functional activity and intrinsic structural properties [38]. The TC 70S particles show a significant difference in the low frequency dispersion as compared to LC ribosomes (Fig. 10, the arrows point out the differences in the actual relaxation frequencies). In particular, the relaxation occurs at a higher frequency while the dielectric increment is lower. This implies that longer traits of rRNA are exposed in LC 70S and it suggests a decrease of particle compactness. Therefore,
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Fig. 10. Permittivity ε vs. frequency of 70S LC ribosomes (upper panel) and TC ribosomes (lower panel). The insets report dielectric loss ε . Arrows visualize the relaxation frequencies of the different samples. Particle concentration was 10 mg/ml.
Fig. 11. Permittivity ε vs. frequency of 30S LC subunits (upper panel) and 30S TC subunits (lower panel). The insets report dielectric loss ε . Arrows visualize the relaxation frequencies. Particle concentration was 10 mg/ml.
we conclude that TC are “denser” than LC particles. This conclusion, obtained by a very simple experimental strategy is in agreement with recent work obtained by solution X-ray and neutron scattering. In this work it was demonstrated that in the 30S the rRNA moiety is more anisometric than the subunit itself. Furthermore, the rRNA of the 50S subunit forms a compact core [39,40]. The same study was performed on the separate subunits obtained from both ribosome species. The 50S TC and LC show the same dielectric behavior and 30S TC resembles 70S since it produces two different dispersions. On the other hand, LC 30S subunits show only the second relaxation but shifted to a lower frequency (Fig. 11). As mentioned earlier, the first dispersion is very likely located out of our measuring window of frequencies [15]. These data are consistent with a “floppier” nature of the LC small ribosomal subunit as compared the homologous TC subunit. In conclusion, the structural differences monitored in the whole ribosome reside mainly in the small subunit. Microcalorimetric measurements on 70S and 50S obtained by the two different protocols, do not exhibit substantial differences in the thermal profile while, as already discussed, the small LC subunit produces an unstructured thermal profile. Particles from TC preparations exhibit, on the contrary, a well-defined peak that indicates a single denaturation event. In addition, the first denaturation peak in LC 70S occurs at a slightly lower temperature; this shows that the intrinsic floppiness of LC 30S affects the overall structure of the ribosome, in agreement with the dielectric data. Functional and structural transitions from TC to LC and vice versa were reported even though no important structural differences were evidenced [41]. An overall conclusion can
be drawn from these data: in TC particles the rRNA is less exposed than in LC particles; furthermore TC ribosomes have a more compact structure. Therefore, the loss of compactness observed in LC ribosomes may be related to their decrease of function in vitro. We investigated the configuration of both ribosome species prior to complete particle collapse. The strategy of these experiments is analogous to the one described previously on LC ribosomes. Also in this case the combination of the three biophysical approaches was adopted. Dynamic light scattering data show that native TC particles are found over a relatively narrow range of diffusion coefficients. About 30% of the whole population, accounts for 70S ribosomes: we remind that in the case of LC ribosomes 20% constituted the monomeric particle species. After the thermal treatment TCs’ form two major groups of particles. The first has a very low value of diffusion coefficient (D) and thus represents aggregates. The second one remains in a range of D that characterizes the native TC 70S and represents about 15% of the total (Fig. 12). On the contrary, as reported above, the thermal treatment of LC couples did not evidence a discrete particle population. A quantitative estimation in this case is impossible, since the thermal treatment produces significant alterations and therefore a form factor should be taken into account. Results of light scattering and dielectric spectroscopy measurements are totally consistent. The higher exposure of rRNA traits after thermal treatment is demonstrated by the increase of the subunit length b which is more relevant for LC than TC particles. The persistence of the second dielectric dispersion suggests a particular stability of the kernel in these latter particles (Figs. 8 and 13 and Table 3
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Fig. 12. Distribution of ribosomal particles as a function of the translational diffusion coefficient. Lower and upper panels show the native and thermally treated TC particles (4 min at 70 ◦ C). Ribosome concentration was 1.8 mg/ml corresponding to about 1013 particles/ml.
which summarizes all data concerning LC and TC 70S particles). Fluorescence measurements show that the second weak binding of EB is essentially the same for both thermally treated and untreated ribosome as far as TC particles is concerning. The number of strong binding sites increases after the treatment but this phenomenon is much less relevant for TC than for LC ribosomes (Figs. 7 and 14 and Tables 2 and 4 which summarize the data on LC and TC ribosomes). This further validates the notion that loose couples are more sensitive to the thermal treatment. The data conclusively demonstrate that the higher functional in vitro activity of TC ribosomes can be associated to intrinsic structural differences that imply a more resistant kernel and a reduced exposure of RNA to the solvent. The ribosomal machinery is obviously very complex and its fundamental role in protein synthesis is well established. Ribosomes are highly conserved throughout evolution and contain large amounts of RNA: more that 50% of the whole mass. Ribosomal RNA is directly involved in translation and its ribozyme role has been suggested. Also ribosomal RNA may be a molecular fossil dating back to the age of early molecular evolution when the RNA had a genetic and catalytic function [42–45]. The data discussed in this work seem to support this idea. However, it should be taken into account that the ribozyme role is still a matter of discussion [46,47]. 2.5. A concise overview of spectroscopic studies on ribosomes present in literature
Fig. 13. Permittivity ε vs. frequency of 70S TC ribosomes. In the inset dielectric loss ε . () Native particles; () and () thermal treatment at 70 ◦ C for 2 and 4 min, respectively. Particle concentration was 10 mg/ml.
A very large number of investigations on ribosomes by spectroscopic approaches, encompassing a great variety of experimental technologies, are present in literature. These studies were conducted on biological macromolecules and also on supramolecular structures as complex as ribosomes. It is therefore very difficult to take into account the abundant literature and to give a general panorama of the results. For instance, in their pioneering works Beychok and Tinoco [48,49] used optical rotatory dispersion and circular
Table 3 Dielectric parameters of the two dispersions observed in native and thermally treated (70 ◦ C) LC and TC particles ε1
ε2
f2 (106 Hz)
b (nm)
70S LC Native 8 ± 1 5.1 ± 0.4 2 min treatment 11 ± 1 4.2 ± 0.4 4 min treatment 19 ± 2 2.5 ± 0.3
13 ± 2 16 ± 2 26 ± 3
3.4 ± 0.6 2.8 ± 0.5 1.3 ± 0.3
73 ± 2 80 ± 3 104 ± 5
70S TC Native 5 ± 2 7.1 ± 0.6 2 min treatment 5 ± 2 7.2 ± 0.7 4 min treatment 15 ± 2 5.8 ± 0.3
16 ± 2 18 ± 3 19 ± 4
2.2 ± 0.6 2.0 ± 0.6 1.8 ± 0.6
62 ± 2 62 ± 3 69 ± 2
f1 (105 Hz)
The last column reports the estimate of the subunit length b. Ribosome concentration was 10 mg/ml.
Fig. 14. Fluorescence measurements on TC particles. Binding isotherms: () native particles; () and () thermal treatment at 70 ◦ C for 2 and 4 min, respectively. The continuous lines are the result of a best fit based on the hypothesis of two different bindings fluorophore/ribosome. Ethidium bromide was at the fixed concentration 5 × 10−7 M. The concentration of ribosomes was varied up to a maximum of 0.1 mg/ml.
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Table 4 Number of sites excluded per mole of bound EthBr
70S TC Native 2 min treatment 4 min treatment
N1 (10−3 )
K1 (107 M−1 )
N2 (10−3 )
K2 (105 M−1 )
1.59 ± 0.05 1.85 ± 0.09 2.20 ± 0.10
1.00 ± 0.05 0.80 ± 0.09 0.80 ± 0.10
5.48 ± 0.06 4.20 ± 0.08 3.10 ± 0.20
5.00 ± 0.07 5.00 ± 0.10 5.00 ± 0.20
The association constants of the strong (N1 , K1 ) and weak (N2 , K2 ) binding for native and thermally treated ribosomes are also shown. Data refer to TC ribosomes.
dichroism to investigate the structure of biopolymers as early as 1968 and 1970; also small angle X-ray dispersion, that permitted the elucidation of DNA structure, was applied to the study of solutions of biological supra-molecular objects [50]. Fluctuation spectroscopy, a more exotic investigation tool, found also a role in molecular biology [51]. We shall cite here some relevant and significant reviews reporting data that we consider more directly related to our studies. This summary, of course, will not be exhaustive. The seminal review “Components of bacterial ribosomes” by the late Prof. Heinz Gunther Wittmann can be a starting point of a review on the structure of the bacterial ribosome and the macromolecules that contribute to its function and structure [52]. Fluorescence measurements were also used in the investigation of the ribosomal structure and function: in particular, five different approaches were adopted, i.e.: emission intensity; emission wavelength maximum; fluorescence anisotropy; collisional quenching; and energy transfer. The results of these investigations allowed a description of the movement and conformation of the main biopolymers (tRNA, nascent peptide, and mRNA) involved in the formation of the peptide bond and in the polypeptide elongation [53]. Furthermore, Xray diffraction, NMR spectroscopy and electron microscopy gave an insight on the complex structure of the rRNA: for instance, ribosomal proteins L11 and S15 show that RNA/protein interactions are involved in the establishment of the ribosome structure at the junctions between ribosomal RNA helices [54–56]. The exchange-transferred nuclear Overhauser effect of NMR spectroscopy may be informative of the interaction between small ligand molecules and high-molecular-weight proteins or nucleic acids. Further applications of the method involve systems of great complexity, such as ribosomes [57]. Electron cryo-microscopy, although strictly speaking cannot be considered a spectroscopic technique, has assumed a growing importance in recent years. Many molecular structures of both prokaryotic and eukaryotic ribosomal complexes have been elucidated by this technique. In the case of E. coli 70S particle a resolution ˚ was reached, while in the case of the extremophyle of 11.5 A ˚ Thermus thermophylus the resolution was even higher (5.5 A) [42]. This allows the identification of structures as minute as RNA helices, peripheral proteins, and intersubunit bridges. Furthermore, analysis of the isolated ribosomal large subunit ˚ However, this 50S, was performed at a resolution of 7.5 A. is yet not sufficient to visualize the amino acid side chains [58,59]. Very recent developments of mass spectrometry
allow the study of protein nucleic acids interactions: in particular the stoichiometry of protein/nucleic acid interactions can be evaluated. This approach can be applied to complex macromolecular machines such as ribosomes [60]. A very high resolution was obtained by X-ray crystallography on ribosomes from mesophilic bacteria and from extremophiles; these works described the structure of the ribosome in various functional states [61–68]. A final consideration is that all the experimental investigations discussed so far, often are highly sophisticated and the preparation of the sample follows complex and costly protocols. It is therefore surprising that using our standard techniques, that complement perfectly each other, allowed us to do interesting observations on the structural properties of the ribosome and their phenomenological correlation to ribosome function.
Acknowledgements The authors are indebted to Cesare Cametti, Knud H. Nierhaus, Giuseppe Onori and Maria Grazia Ortore for their constant interest and active collaboration in the development of this research. The subjects covered in this review were included in the master degrees of several students whose valuable cooperation is also acknowledged.
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