Faculty of Physics and Faculty of Chemistry1, Department of Virology and A. N. Belozersky Institute of Physico-Chemical Biology2, Moscow State University, Vorobiovy Gory, Moscow 119899, Russia
Author for correspondence: Josef Atabekov. Fax +7 095 938 06 01. e-mail Atabekov{at}genebee.msu.su
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Abstract |
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Introduction |
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Atomic force microscopy (AFM) (Binnig et al., 1986 ) is a high-resolution technique which allows visualization of proteins (Kiselyova & Yaminsky, 1999
), nucleic acids and nucleoprotein complexes (Drygin et al., 1998
; Lyubchenko et al., 1993
, 1995
; Valle et al., 1996
; Shlyakhtenko et al., 1998
; Yodh et al., 1999
; Jafri et al., 1999
). To our knowledge this work presents the first report of examination of the TMV MPRNA RNP complexes by means of AFM.
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Methods |
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Expression and purification of (His)6-tagged TMV MP.
Expression in Escherichia coli and purification of the 30 kDa (His)6-tagged TMV MP was carried out as described by Karpova et al. (1997) . The purity of all the MP preparations isolated from E. coli by metal chelate affinity chromatography was verified by SDSPAGE.
TMV MPRNA complex formation.
MP in twice-distilled water (0·1 µg/µl) was incubated with TMV RNA or short synthetic RNA transcripts on ice for 5 min at different molar MP:RNA ratios. In some cases the complexes were treated with RNase A (20 min at room temperature with 0·5 µg of RNase). RNA transcripts were obtained by in vitro transcription with T7 polymerase (Ribomax kit, Promega) of the linearized plasmid that contained an 890 nt artificial construct. The construct was cloned into pBluescript SK+ vector (Stratagene).
AFM measurements.
For AFM measurements, the preparations were deposited onto substrates of freshly cleaved mica, incubated for 5 min, rinsed with distilled water and dried in airflow. Observations were with a Nanoscope III multimode scanning probe microscope (Digital Instruments). Standard silicon 125 µm cantilevers (NanoProbe) with 300350 kHz resonant frequency were used in all the experiments carried out in tapping mode. For image processing, user-friendly software Femtoscan 001 (Filonov & Yaminsky, 1997 ) was employed.
Sedimentation analyses.
These were done in a Spinco Model E analytical ultracentrifuge equipped with absorption optics.
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Results and Discussion |
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MPTMV RNA complexes formed at different molar MP:RNA ratios were visualized by AFM. A mica surface is negatively charged in distilled water, whereas the MP is positively charged under these conditions. Therefore, we used freshly cleaved unmodified mica for imaging the MPRNA complexes. In the first experiments the complexes produced at a molar MP:TMV RNA ratio of 20:1 (corresponding to 320 nt per MP molecule) were examined. In a previous study of TMV MPRNA complex formation using a nitrocellulose membrane filter binding assay it was reported that at an MP:RNA molar ratio of 25:1 virtually all the RNA remained unbound, whereas at a ratio of 50:1 about 60% of the TMV RNA was involved in complex formation (Karpova et al., 1997 ). However, we show here by AFM that some MPRNA complexes were formed even at a molar MP:RNA ratio as low as 20:1 (Fig. 1a
, b
). The structures presented in Fig. 1
are referred to as beads-on-a-string, with the apparent dimensions of the beads identical to those of individual MP molecules in control MP preparations. Fig. 1
shows that the MP molecules are distributed along the RNA chain with detectable minimal distances between the centres of two vicinal resolved globules (beads) of about 15 nm, although the distance between the neighbour globules varied over a wide range. Apparently, the MP molecules observed as vicinal beads in this type of MPRNA complex are in fact separated by protein-free RNA segments of about 12 nm (Fig. 1a
, b
). The majority of the globules appear to represent individual MP molecules bound to RNA independently of each other and separated by protein-free RNA segments of varying length (Fig. 1c
). Not infrequently these RNA segments could be revealed by AFM (arrows in Fig. 1a
, b
). These results imply that at a molar MP:RNA ratio as low as 20:1 the binding of MP to RNA is not cooperative, i.e. the MP molecules do not interact with each other upon RNP complex formation. The larger globules (presumably dimers and/or trimers) made up not more than 30% of the total amount of RNA-bound MP. Individual TMV RNA molecules free of MP could not be detected in these samples under conditions used to visualize MPRNA complexes since a special cationic treatment of the mica surface is needed for RNA immobilization. Thus, the presence of MP-free RNA molecules in the MPRNA mixture is very likely.
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Next, AFM was used to characterize MP distribution along TMV RNA within complexes formed at MP:RNA ratios of 60:1 and 100:1 (corresponding to 100 and 60 nt per MP molecule, respectively). Large globular particles (about 40 nm in height and 150200 nm in diameter) with unresolved structure were revealed in AFM images after the MPRNA mixture was incubated on ice (data not shown). However, the globules formed at the molar ratio of 60:1 could be unfolded by 40 min incubation at room temperature, producing linear structures with several bends and approximately constant lateral dimensions (Fig. 2a, b
). This type of complex will be referred to as a thick string particle. Unlike the beads-on-a-string formed at a molar ratio of 20:1 the MP monomers are evidently packed more densely in the thick string. The length of thick string complexes could not be determined precisely because of their tendency to form linear aggregates. However, the apparent width of the complexes measured at half height (about 1518 nm) exceeded that of a single MP molecule (1012 nm) and the height of the complex (2·53·5 nm) was several times that of protein-free RNA (i.e. 0·30·5 nm, according to Drygin et al., 1998
) and 1·52·0 times higher than that of individual MP molecules. In most images quasi-periodic variations of height were observed within the structure of the thick string complexes (Fig. 2a
, b
), suggesting that they consist of tightly packed blocks, as clearly seen on the cross-section of the AFM image (Fig. 2c
). It should be emphasized that the distance between the centres of blocks varied greatly (from 22 to 50 nm). Therefore, the period of 25·5 nm revealed in Fig. 2(c)
is not a universal parameter of thick string complexes, but characterizes only the dimensions of cross-section made along a particular randomly selected region of the complex. The segments with constant height and width (marked by arrows in Fig. 2a
) might consist of more densely packed molecules which were not resolved by AFM. The presence of bends in the thick string complexes (Fig. 2
) can be explained by taking into account the fact that an AFM image is a two-dimensional projection of a three-dimensional conformation of the complex. Presumably, the segments densely covered with MP molecules acquire rigidity and are seen as practically straight lines. Most probably, bending occurs at sites where RNA is free of MP.
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In further experiments, short (890 nt) synthetic RNA transcripts were used instead of TMV RNA in order to examine the structure of short MPRNA complexes produced at non-saturating MP:RNA ratios corresponding to 80 nt, 30 nt and 20 nt per MP molecule (molar MP:RNA ratios of 10:1, 30:1 and 40:1, respectively). According to Citovsky et al. (1990) , most RNA transcripts should be fully coated with MP under such conditions, although some molecules remain entirely free of protein and, as was mentioned above, cannot be visualized by AFM under the conditions used. Fig. 3(a
c
) shows the complexes formed by the 890 nt transcripts with TMV MP. The mean length of these complexes was about 350 nm, suggesting that they consist of about 130 individual MP molecules with average lateral dimensions of about 3 nm (see above). According to Citovsky et al. (1990)
, each MP molecule binds 7 nt in the unfolded MPRNA complex produced in vitro, which is consistent with a packing density of about 130 MP molecules per 890 nt. It is worth mentioning that the structure of the complexes produced at all three molar MP:RNA ratios was identical and indistinguishable from that of thick string-type complexes described above, i.e. the centre-to-centre distance (2250 nm) between the blocks, the height (2·53·5 nm) and the diameter measured at half height (1518 nm) corresponded to the parameters of the thick strings formed by the MP and TMV RNA (Fig. 2
). It is important to note that these complexes were resistant to RNase attack (not shown). It can thus be suggested that the RNP particles produced by short RNA transcripts and MP structurally mimic the clusters of densely packed MP molecules cooperatively bound to discrete regions of full-length TMV RNA in the thick strings. Interestingly, we did not detect complexes of the beads-on-a-string type in experiments with short RNA transcripts.
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It is believed that MPTMV RNA complexes may represent cell-to-cell intermediates (transport form) of virus infection (for recent review, see Tzfira et al., 2000 ). It is widely accepted that TMV MP binding to RNA is cooperative, i.e. the MP molecules interact with each other upon RNP formation. The results of AFM analysis taken together with the results of nitrocellulose membrane filter binding assays (Karpova et al., 1999
) indicate that at subsaturating MP concentrations at molar MP:RNA ratios of 60100:1 only clusters of MP molecules can be bound cooperatively to discrete regions of RNA, producing the thick string structures. The MP molecules in thick string particles are packed tightly enough to protect RNA from RNase attack. It is tempting to suggest that the MPRNA complexes produced in vivo are formed under saturating MP concentrations and therefore represent a continuous extended RNP with the thick string structure.
The real dimensions of unfolded MPRNA visualized by AFM (height of the complex 2·53·5 nm) and by electron microscopy (1·52·5 nm in diameter) (Citovsky et al., 1992 ) are of the same order of magnitude. Taking into account that the permeability of the plasmodesmata is increased by TMV MP from 0·751·0 kDa to 20·0 kDa (Wolf et al., 1989
; Waigmann et al., 1994
), the modified plasmodesmata correspond to a dilated channel diameter of 59 nm. In other words, the extended complexes with a diameter between 2·5 and 3·5 nm are potentially suitable for trafficking through the MP-modified plasmodesmata.
Recent studies indicate that TMV MP and/or MPRNA complexes interact with cytoskeleton elements (Heinlein et al., 1995 ; McLean et al., 1995
) and with the endoplasmic reticulum (Heinlein et al., 1998
). Consequently, both intracellular and cell-to-cell translocation of the viral genome may involve the interaction of MPRNA complexes with different cellular structures, including cytoskeleton elements, endoplasmic reticulum and plasmodesmata.
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Acknowledgments |
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References |
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Received 29 November 2000;
accepted 1 February 2001.