Vol.
178,
August
No. 15,
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Pages 1280-1287
1991
SCANNING
TUNNELLING
OF 16s RIBOSOMAL
MICROSCOPY RNA IN WATER
Eric Lesniewska,Pierre-JacquesFlamion*, Claire Cachiaand Jean-PierreSchreiber Laboratoire de Biophysique, Faculte de Pharmacie,7 Boulevard Jeanned’Arc, 21000Dijon, France and Jean-PierreGoudonnet Laboratoire de Physique du Solide (U.R.A. 785 CNRS), FacultCdes SciencesMirande, 21004Dijon, France Received
May
13,
1991
SUMMARY:
The scanningtunnelling microscopehasbeen usedto image 16s ribosomal RNA moleculesin water electrophoretically depositedon graphite surface. Two kinds of imageshave been obtained: images showing aggregatesof 16s ribosomal RNA molecules similar to those obtained from DNA solutions and others showing individual 16s ribosomal RNA molecules.An interesting characteristic of these images, recorded in constant current mode, is that the 16s ribosomal RNA moleculesappearto be located below the graphite surface. The morphology and several structural parametersof the moleculeswere consistentwith the data obtainedfrom electron microscopy. e 1991Academic Press,Inc. An important characteristic of the STM is its ability to image topographic profiles or electronic structureswith an atomic or near-atomicresolution (l-3). Numerousbiological moleculessuch as nucleic acidsor proteins and more complicatedbiological structuressuchasvirus were imaged by STM (4-7, 8). Most of these works have been performed under atmospheric conditions by observing air-dried samples.Under theseconditions, the secondary and tertiary structuresof the moleculesare certainly perturbedmainly by dehydration and the influence of the ionic environment of the solution on the structuralpropertiesis ruled out. The possibility of operating,with atomic resolution, an STM in liquids and in various buffers offers a new way to study three dimensionalstructuresof macromoleculesin solution (9, 10). The 16s rRNA of the small subunitof E.coli ribosomeis an interestingmolecule to be imaged by STM. The secondary structure has been entirely described (11) and numerous studies have been made to elucidate the tertiary structure (for reviews, seerefs. 12, 13) which leaded to the elaboration of various three-dimensionalmodels(14, 15). The folding of free 16s r-RNA dependsstrongly on the * To whom requestsfor reprints shouldbe addressed. The abbreviationsusedare: 16srRNA, 16s ribosomalRNA E. coli, Escherichiacoli; STM, scanningtunnelling microscope(y);EM, electronmicroscope(y); STEM, scanningtransmission electron microscope(y);HOPG, highly orderedpyrolitic graphite. 0006-291X/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
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ionic composition of the buffer solution. In reconstitution buffer, 16s rRNA presents a compacted tertiary structure characterized by a radius of ,Tration of X&10 A (16, 17). From samples prepared in water, EM and STEM studies showed that 16s rRNA appears as an extended molecule with a simpler structure than in usual reconstitution buffers (18). Therefore, the description of the conformation of 16s rRNA in water is an interesting first step for the study of its folding under various buffer conditions. With an STM operating under atmospheric conditions, we imaged recently air-dried 16s rRNA molecules, prepared from compacting buffer conditions (19). V-shaped molecules were seen and we showed from STM images that structural parameters such as volume or radius of gyration could be determined. We report in this paper the fiit STM images of 16s rRNA molecules in water and distributed on graphite surface by electrophoretic deposition.
MATERIALS
AND
METHODS
16s i-RNA: The 30s subunits of E.coli MRE 600 ribosomes were isolated by zonal sucrose gradient centrifugation (20). 16s rRNA was prepared according to the method described previously by Cachia & al. (21) and stored as a precipitate in ethanol-buffer A (50 mM ammonium acetate, pH 5.6) mixture (2:l). Sample preparation: 16s rRNA was pelleted by centrifugation at 10000 * g for 10 min and the pellet was dissolved in water (Milli-Q 18 ML!). Then, this solution was diluted in order to obtain a concentration of 2.5 lo-10 M (15 1010 molecules / ml) monitored by spectrophotometry. Absorbance spectra were recorded on a Varian Cary 2200 spectrophotometer and the molar absorptivity of the 16s rRNA at 260 nm was taken as 1.25 107 M-l cm-l (22). Scanning: Tunneling Microscopv: The tunneling microscope used for this study is a built-in airbased STM (23) with the main following characteristics. The tunneling current (ranging from 1OpA to 10 nA) is measured by using an amplifier Keithley 427 (Keithley Instrument, Cleveland, OH). The piezoelectric tube (PZT-8H from Channel Indus Santa Barbara, CA) had a sensitivity of 4.44 rim/V for lateral displacements (resolution of 0.2K j and 3.4 rim/V for vertical displacements (resolution of 0.05 A). The z motion was calibrated on gold substrates. On freshly cleaved mica, an atomic flat gold surface can be evaporated with an interplane spacing of 2.354 A which allows the z calibration. The tunneling tips were mechanically sharpened from 0.25 mm Pto7-Iro.3 wire. The tips were insulated with molten Apiezon wax (Angstrom Technology, Mesa, AZ) as indicated previously (10). The electrochemical cell was equipped with a platinum loop as counter electrode and a reference electrode to monitor the electrodeposition. A current amplifier allowed the working electrode to remain at virtual ground and to obtain an information directly proportional to the current in the electrochemical cell. Usually, a deposition voltage of -2V was applied between the counter electrode and the substrate during 3-5 min to electrodeposit the 16s rRNA molecules. mlitative and auantitative analvsis of STM images: The images consist of 100x100 or 128x128 z(x,y) data points representing the surface topography of a scanning area. Samples were scanned at a speed ranging from 50 run/s to 500 rim/s simultaneously in the constant-current and voltagemodulated modes. In the standard topographic mode, the tunnelling current is maintained at a fixed value of 1 nA by the feedback loop. Simultaneously, the bias voltage (- 50 mV) was modulated with a small ac voltage to determine AIIAV. As a matter of fact, the quantity AI/AV is roughly proportional to the local single electron density of states of the scanned material in the low-voltage limit (24). This spectroscopic mode can help to distinguish graphite from other material deposited on the HOPG (Union Carbide, Cleveland, OH) substrate surface (19,25). The structural parameters of macromolecules such as length, width and height, were determined from line scan profiles. Area, volume and radius of gyration of the structures were calculated from regions of the STM image selected by a box generated from the computer keyboard as described 1281
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previously (19). All the data points of this region with a z value lower than the ~0 background are consideredasbelongingto the particle.
RESULTS The electrochemicalcell wastestedby imagingthe HOFG surfacein water with tips testedbefore on HOFG in air. We obtained atomically resolved imageswhere the graphite appearedas a regular lattice with no apparentdefects. Theseimageswere stableduring continuous scanningsand they showed a corrugation amplitude of 2 8, consistent with the calculations. No structures such as it appearsfor the 16s rRNA solutionswere observed. The deposit of 16s rRNA moleculeson the HOPG surface was performed by using the method describedby Lindsay et al. (10,26) with somemodifications. 200 l.rl of 16s rRNA solution with a concentration of about 2.5 10-m M were introduced in the cell. The required concentration of the solutionwasroughly estimatedfrom the equation:
‘=NS;EAt
where n is the averagenumberof moleculesexpected in the squarescanningarea S (1000 A x 1000 A), N the Avogadro’s number,p the mobility (108m2 s-r V-l), E the electric field (103 V/m) andAt (3 min) the duration of the electrodepositionwhich gives a concentrationC of about 2.5 lo-10 M. A voltage of -2V was applied between the counter electrode and the substrateto electrodeposit the molecules.The backgroundcurrent wassmallerthan 10 PA. After an electrodepositionperiod of 35 min, the solution wasremoved in order to eliminate the non-adsorbedmoleculesandreplaced by 200 p,l of water. The tunnelling approachof the tip was then performed in water. The imagesof the observing areawere comparedbefore and after the electrodepositionstep.We can considerthat the re-positionning of the tip on the sameplace after a z piezo displacementof 1000 A ( to allow the electrodepositionand the fluid change), was reproducible with an accuracy of 10%. The deposits which appearedduring the electrodepositionstepwere consideredas 16s rRNA molecules. In Figure 1, we showa typical imageof aggregatesof 16s rRNA moleculesin water. In this figure, 16s rRNA molecules appear as long parallel rod-like molecules similar to the DNA molecules obtained by Lindsay et al. (10, 26) with rods of 42 f 11 8, (n = 23) in diameter. We obtained numerous images as those shown in Fig.1 which were difficult to use for structural studies. Fortunately, we obtained also, but more rarely, imagessuch as those shown in Fig. 2. Fig. 2(a) representsan individual 16s rRNA moleculenearly complete (scanningarea 1000 x 1000A) and Fig. 2(b) line scandata. The geometrical arrangementof the molecule strandsreveals a strong interaction betweenthe surfaceand the moleculeprobably trappedby graphite stepswhich were not viewed during our preliminary observation.In this figure, the 16s rRNA moleculeis characterized by a length larger than 1600 A (and a radius of gyration upper than 325 A). From imagessuchas Fig. 2, we determined an average backbone width of 4.5k 8 A. Figures 3 (a) and 3 (b) show 1282
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STh4 images of aggregates of 16s rRNA on HOPG surface in water. Fieldwidtb = 1000
respectively an enlargement of the protrusions clearly visible in Fig. 2 (a) and line scan data. These protrusions
are similar to the principal ones observed by Mandiyan et al. (18) but often differ in
length and shape. The average width of the protrusions
(25 f 5 A) suggests a number of strands
less important that it was indicated from STEM data. In Table I, we compare the main structural parameters obtained from STEM and STM studies. These results are in good agreement except for the length (more than 1600 A instead of 1200 A) and the number of strands in the major protrusion. It is interesting to note that for all the images of 16s rRNA obtained in water the tip dipped down over the molecule and therefore, the z-displacements
representing the topographic surface AZ =
f(x,y) were lower than the one of the graphite surface. Note also the large values of the AZ displacements ( 30-40 8, ) shown in the cross-sections ( Figs. 2(b) and 3(b) ) over the molecules .
DISCUSSION One of the aims assigned to STM studies on biological molecules is to sequence DNA molecules. STM can also be considered as an interesting tool for structural studies of nucleic acids and its ability of operating in solution is an important advantage compared to the electron microscopic techniques. But at present, few results obtained under these conditions are available. Because STM is a new high resolution microscopy, numerous artefacts that sometimes look like nucleic acid molecules on HOPG substrates, can be observed and misinterpreted (27, 28). 1283
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b r
100
0
X(nm)
Figure 2 . (a) STM image of individual 16s rRNA molecule. Fieldwidth = loo0 A. (b) Line scan data showing a backbone width of 38 A and a negative z-displacement of 35 A.
Therefore, comparisons between several kinds of experiments are recommended. We used the deposition technique of Lindsay et al. (10, 26) and we found that the main statistical and physical results they obtained from DNA molecules were also valid for 16s rRNA. In particular, the long rod-like molecules as shown in Fig. 1 were easily imaged but individual molecules as presented in Fig.2 were more difficult to obtain. We observed also that the tip dipped down with the same AZ values ( 10-50 A) over the 16s rRNA molecules in order to maintain the tunnelling current constant. These large negative AZ values proove that what it is imaged as substrate is not the graphite surface 1284
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o-
Im 1
I -4 __ 0
X(-l
40
Fieure3 . (a) STM imageof the same16srRNA molecule. Fieldwidth= 400A. (b) Line scan datashowin a protrusionwidth of 21A, a backbone width of 358, anda negativez-displacement closeto 35x.
but probably a wide layer of small adsorbed molecules, that seemsto confirm entirely the observationsand the explanationsof the contrast mechanismdeveloppedpreviously and basedon the adsorbate deformation (10, 26, 29). The formation of this layer can be attributed to the electrodepositionstepbecauseit was impossibleto obtain the atomic resolutionon the graphite after this phase. Structural parametersof the moleculeproove alsothat what it is viewed is not an artefact. For DNA studies,helix pitch, height, minor and major groove width are parametersthat help to interpret the 1285
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some of the JM105
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COMMUNICATIONS
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Vol.
178, No. 3, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
11. Gutell, R.R., Weiser, M., Woese, C.R. & Noller, H.F. (1985) Progr. Nucl. Acids Res. Mol. Biol. 32, 155-216. 12. Hill, W.E., Moore, P.B., Dahlberg, A., Schlessinger, D., Garrett, R.A. and Warner J.R. (Eds.) (1990). The t&some: Structure, Function & Evolution, Springer-Verlag, New York. 13. Noller, H.F., Moldave, K. @is.) (1988), Methods Enzymol. 164, Academic Press, New York. 14. Brimacombe, R., Atmadja, J.; Stiege, W. 8z Schuler, D. (1988) J. Mol. Biol. 199, 115136. 15. Stern, S., Weiser, B. & Noller, H.F. (1988) J. Mol. Biol. 204, 447-481. 16. Boublik, M., Oostergetel, G.T., Mandiyan, V., Hainfeld, J.F. & Wall, J.J. (1988) Methods Enzymol. 164,49-63. 17. Mandiyan, V., Tumminia, S., Wall, J.S., Hainfeld, J.F. & Boublik, M. (1989) J. Mol. Biol. 210, 323-336. 18. Mandiyan, V., Hainfeld, J.F., Wall, J.S. & Boublik, M. (1988) FEBS Lett. 236, 340-344. 19. Flamion, P.-J., Cachia, C., Schreiber, J.-P., David, T., Lesniewska, E. & Goudonnet, J.-P. J. Microsc. (in press). 20. Hardy, S.J.S., Kurland, C.G., Voynow, P. & Mora, G. (1969) Biochemistry 8, 2897-2905. 21. Cachia, C., Flamion, P.J. & Schreiber, J.P. (1990) J. Chromatogr. 498, 417-422. 22. Serdyuk, I.N., Agalarov, S.C., Sedelnikova, S.E., Spit-in, A.S. & May, R.P. (1983) J. Mol. Biol. 169, 409-425. 23. Becker, R.S., Golovchenko, J.A. & Swartzentruber, B.S. (1985) Phys. Rev. Lett. 54, 26782680. 24. Kuk, Y. & Silverman, P.J. (1989) Rev. Sci. Instrum. 60, 165-180. 25. Allison, D.P., Thompson, J.R., Jacobson, K.B., Warmack & R.J., Ferrell, T.L. (1990) Scanning Microsc. 4,5 17-522. 26. Lindsay, S.M., Thundat, T. & Nagahara, L. (1988) J. Microsc. 152,213-220. 27. Salmeron, M., Beebe, T.P., Gdriozola, J., Wilson, T., Ogletree, D.F. & Siekhaus, W. (1990) J. Vat. Sci. Technol. A 8,635&l. 28. Clemmer, C.R. & Beebe, T.P. (1991) Science 251,640~642. 29. Thundat, T., Nagahara, L.A., Oden, P. & Lindsay, S.M. (1990) J. Vat. Sci. Technol. A 8, 645-647. 30. Driscoll, R.J., Youngquist, G. & Baldeschwieler, J.D. (1990) Nature 346, 294-296. 31. Vasiliev, V.D., Koteliansky, V.E., Shatsky, I.N. & Rezapkin, G.V. (1977) FEBS Lett. 84, 43-47. 32. Vasiliev, V.D., Selivanova, O.M. & Koteliansky, V.E. (1978) FEBS Lett. 95, 273-276.
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