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From the
Received for publication, June 8, 2000, and in revised form, July 13, 2000
The shape of free Thermus flavus 5 S
rRNA in solution at 1.3 nm resolution is restored from synchrotron
x-ray scattering data using an ab initio simulated
annealing algorithm. The free 5 S rRNA is a bent elongated molecule
displaying a compact central region and two projecting arms, similar to
those of the tRNA. The atomic models of the 5 S rRNA domains A-D-E and
B-C in the form of elongated helices can be well accommodated within
the shape, yielding a tentative model of the structure of the free 5 S
rRNA in solution. Its comparison with the recent protein-RNA map in the
ribosome (Svergun, D. I., and Nierhaus, K. H. (2000) J. Biol. Chem. 275, 14432-14439) indicates that the 5 S rRNA
becomes essentially more compact upon complex formation with specific ribosomal proteins. A conceivable conformational change involves rotation of the B-C domain toward the A-D-E domain. The model of free 5 S rRNA displays no interactions between domains E and C, but such
interactions are possible in the bound molecule.
Ribosomal 5 S rRNA, an essential component of the ribosome, is
approximately 120 nucleotides long. The ribosomal particles lacking the
5 S rRNA have a strongly reduced activity in protein synthesis (1-3),
in particular, reduced peptidyl transferase activity. Because of its
functional importance and the fact that the 5 S rRNA interacts
specifically with several ribosomal proteins (4), it is of great
interest to know the three-dimensional structure or a reliable shape of
this RNA molecule. Nearly 1000 different prokaryotic and eukaryotic 5 S
rRNA sequences have been determined so far. The predicted secondary
structure (5) is shown in Fig. 1. The
size of the 5 S rRNA limits the possibility of its three-dimensional
structure determination by nuclear magnetic resonance (NMR).
Therefore, in the past, we tried to crystallize several 5 S rRNA
species (6, 7). The crystals of an isolated 5 S rRNA suitable for the
x-ray analysis were obtained particularly from the thermophilic
bacterium Thermus flavus. These crystals, however, diffract
only to about 0.75 nm resolution and are extremely sensitive to
radiation, even under cryogenic conditions. The intrinsic flexibility
of the whole 5 S rRNA molecule and small differences in the primary
structure seem to influence significantly the quality of the crystals.
The T. flavus 5 S rRNA was then divided into five domains, A
through E, and these domains were chemically synthesized and
crystallized for x-ray analysis (8, 9). In parallel, we began to
analyze the shape of the entire 5 S rRNA in solution using small angle
scattering, an established method of monitoring the low resolution
structure of biological macromolecules (10). Recently, new approaches
have been developed to ab initio restore low resolution
structure from the scattering data (11, 12), and these were
successfully applied to study proteins and ribosomes (13-15). In the
present paper, an ab initio low resolution model of
the free 5 S rRNA in solution is derived from synchrotron radiation small angle x-ray scattering
(SAXS)1 data. The atomic
models of the 5 S rRNA fragments are tentatively positioned inside the
low resolution shape. The model obtained may be further used for
crystallographic molecular replacement studies, as well as for the
analysis of the complex formation with binding proteins in
solution.
Isolation and Purification--
The Escherichia coli
cells from strain MRE600 were grown at 37 °C in a Luria broth
medium. The 70 S ribosomes were isolated by previously described
extraction and centrifugation steps (2). The intact ribosomes were
dissociated into their 30 and 50 S subunits at magnesium concentrations
below 5 mM (1). The 5 S rRNA was separated from the large
ribosomal subunit by saccharose gradient, phenol extraction and
subsequently purified by Sephadex G150 gel chromatography as well as
hydrophobic affinity interaction chromatography. In addition T. flavus 5 S rRNA was produced by in vitro transcription. Because of large yields of up to 5 mg/ml, sufficient RNA material can
be generated at low cost and within a short time compared with the
conventional isolation procedure. The reconstitution of ribosomal
proteins from Bacillus stearothermophilus with 5 S rRNA of
different species resulted in functionally intact 50 S subunits active
in polypeptide synthesis (3). This finding emphasizes that the
three-dimensional structure of the ribosomal 5 S RNA is highly
conserved, making it possible to replace the 5 S rRNA from E. coli by that from T. flavus. Two synthetic DNA oligonucleotides coding for the 5' and 3' regions of T. flavus 5 S rRNA with an overlapping area of 25 bases were annealed
by stepwise cooling from 70 to 54 °C within 17 min. A
double-stranded template was generated by elongation with
Pfu polymerase for 10 min. Restriction sites and T7 promotor
were introduced by primers used in the following polymerase chain
reaction. The product was blunt end-ligated into the vector
pUC18 (2686 base pair) and transformed into E. coli JM109
cells sensitive to blue/white screening. The correct sequence of the
insert was checked by sequencing the isolated plasmids from several
clones. After plasmid linearization with EcoRV (NEB), the
wild-type 5 S rRNA of T. flavus was synthesized during run
off transcription in the RiboMAX system (Promega). The RNA was purified
by phenol extraction and gel filtration. The samples were concentrated
by ethanol precipitation and resuspended in 5 mM
MgCl2 buffer. Purity and homogeneity of the product were analyzed by UV spectroscopy, denaturating urea polyacrylamide gel electrophoresis (6), and dymanic light scattering. Monodispersity of the samples was controlled by dynamic light scattering using a newly
developed experimental set-up (16).
Scattering Experiments and Data Processing--
The SAXS data
were collected using standard procedures on the X33 camera (17-19) of
EMBL at the storage ring DORIS III of the Deutsches Elektronen
Synchrotron (DESY) and multiwire proportional chambers with a delay
line readout (20). The scattering curves at the solute concentrations
0.5, 1, 2, 3, 5, 7, and 10 mg/ml were measured at sample detector
distances of 3.5 and 1.4 m, covering the momentum transfer ranges
0.25 < s < 1.8 nm Shape Determination--
The shape of the 5 S rRNA was restored
from the experimental data using an ab initio method (12). A
sphere of diameter Dmax is filled by a regular
grid of points corresponding to a dense hexagonal packing of small
spheres (dummy atoms) of radius r0 Atomic Models--
The atomic coordinates of the theoretical
model of the 5 S rRNA from Xenopus laevis (26) were taken
from the Protein Data Bank (27), entry code 1rrn. The solution
scattering curve from this model was computed using a modified version
of the program CRYSOL (28). The atomic models of the 5 S rRNA fragments
A, D, and E, available from x-ray crystallography (Refs. 29-31; entry codes 364D, 361D, and 353D, respectively), were used as
templates to fit into the low resolution shape. The models of the 5 S
rRNA were displayed on a SUN SPARC-20ZX work station using the program ASSA (32) and on a SGI O2 work station using the program O
(33).
The apparent radius of gyration of the 5 S RNA in solution
decreases with concentration (Fig.
2a), which is typical for
solutions with repulsive interactions. The value Rg = 3.46 ± 0.05 nm is obtained after extrapolation to infinite
dilution. The composite SAXS curve from the 5 S rRNA extrapolated to
zero concentration is presented in Fig. 2b. The maximum
dimension and the radius of gyration of the particle are 12.0 ± 0.5 and 3.44 ± 0.05 nm, respectively, in good agreement with the
values reported in earlier solution scattering studies (34, 35). The
distance distribution function p(r) in Fig.
2c is typical for an elongated particle. The shape
scattering curve after a constant subtraction (Fig. 2b,
curve 2) yields the Porod volume of 37 ± 2 nm3.
This volume corresponds well to the dry volume of the 5 S RNA (38.8 nm3) computed from its molecular mass, assuming the partial
specific volume of 0.53 cm3/g (36). The above results
confirm that the solutions of the 5 S rRNA are monodisperse and
that the shape scattering curve can be used for the shape analysis.
Ten independent ab initio shape determinations were
performed starting from different random configurations within a
spherical search volume with the diameter
Dmax = 12 nm. Different packing radii of
the dummy atoms were used (r0 = 0.4, 0.35, and 0.32 nm, corresponding to the numbers of dummy atoms
M = 2550, 3660, and 4897, respectively). The
independently restored models had the radius of gyration of 3.49 ± 0.05 nm, volume 36.7 ± 1.0 nm3 and the maximum
dimension 11.7 ± 0.3 nm; they all provided nearly identical fits
to the data illustrated in Fig. 2b (curve 3) with the discrepancy
Structure of Free Thermus flavus 5 S rRNA at 1.3 nm
Resolution from Synchrotron X-ray Solution Scattering*
,,,
,**
Institute of Physiological Chemistry,
University Hospital Hamburg, c/o Deutsches Elektronen Synchrotron,
Building 22a, Notkestra
e 85, 22603 Hamburg, Germany, the
§ Dierks and Partner Systemtechnologie, Pinneberger Weg
22-24, 20257 Hamburg, Germany, the ¶ Institute of Biochemistry,
Freie Universität Berlin, Thielallee 63, 14195 Berlin, Germany,
the ** European Molecular Biology Laboratory, Hamburg Outstation, c/o
Deutsches Elektronen Synchrotron, Notkestrae 85, 22603 Hamburg,
Germany, and the Institute of
Crystallography, Russian Academy of Sciences, Leninsky prospect
59, 117333 Moscow, Russia
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (25K):
[in a new window]
Fig. 1.
Secondary structure of the T. flavus 5 S rRNA according to biochemical and analytical
methods such as enzymatic cleavage, chemical modification, and sequence
alignment studies (5).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 and
0.40 < s < 5.5 nm1,
respectively (s = 4
sin
/
, where 2 is the
scattering angle and = 0.15 nm is the x-ray wavelength). The
data were normalized to the intensity of the incident beam corrected
for the detector response, the scattering of the buffer was subtracted,
and the difference curves were scaled for concentration using the
program SAPOKO.2 The reduced
data sets at low angles were extrapolated to zero concentration
following standard procedures (10). The final composite
scattering curve was obtained by merging the extrapolated low angle
data with the curve recorded at higher angles at 10 mg/ml. The maximum
dimension Dmax of the 5 S rRNA was estimated from the experimental data using the orthogonal expansion program ORTOGNOM (21). The radii of gyration Rg at different concentrations were evaluated using the Guinier approximation (10) and
the indirect Fourier transform program GNOM (22, 23). The latter
program was also used to compute the distance distribution function
p(r). Prior to the shape analysis, a
constant was subtracted from the experimental data to ensure that the
intensity at higher angles decays as
s4 following Porod's law (24) for
homogeneous particles. The value of this constant is determined
automatically by the shape determination program DAMMIN (described
under "Shape Determination") from the outer part of the
curve by drawing a straight line in coordinates s4I(s) versus
s4. This procedure reduces the contribution from scattering
due to the internal particle structure and yields an approximation of
the "shape scattering" curve (i.e. scattering from the
excluded volume of the particle filled by constant density). The Porod volume Vp (24) was calculated from the shape
scattering curve as described (10).
Dmax. The structure of the dummy atoms model
(DAM) is defined by a configuration vector X assigning an
index to each atom (0 corresponds to solvent and 1 to the solute
particle). The scattering intensity I(s) from the
DAM is evaluated as
where the partial amplitudes
Alm(s) are as follows.
(Eq. 1)
Here, the sum runs over the dummy atoms with
Xj = 1 (particle atoms), rj,
(Eq. 2)
j are their polar coordinates, va = (4r03/3)/0.74 is the
displaced volume per dummy atom, jl(x) and Ylm() denote the spherical Bessel
function and the spherical harmonics, respectively. In keeping with the
low resolution of the solution scattering data, the method searches for
a compact interconnected configuration X, minimizing the
discrepancy 
between the calculated and the experimental
curves,
where N is the number of the experimental points and
Iexp(sk) and
(Eq. 3)
(sk) are the experimental intensity after a constant subtraction and its standard deviation in the k-th point, respectively. Starting from a random
configuration, simulated annealing (25) is employed for the
minimization. The details of the program DAMMIN are described elsewhere
(Refs. 12, 14, and 15; see also the EMBL Web page).
RESULTS
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (11K):
[in a new window]
Fig. 2.
a, concentration dependence of the
radius of gyration computed from the scattering by the 5 S rRNA
solutions. b, x-ray scattering from the 5 S rRNA in solution
and scattering calculated from the models. 1, composite
experimental curve; 2, shape scattering curve after
subtraction of a constant; 3, scattering from the ab
initio dummy atom model; 4, scattering from the model
of Westhof et al. (26). c, distance distribution
function of the 5 S rRNA evaluated by the program GNOM.
= 0.42. As the method yields models at an
arbitrary orientation and handedness, the models were
appropriately rotated and shifted for comparison. The overall
appearance of the models displayed in Fig.
3a for five restorations was
very similar; all of them were bent
elongated particles with a
cross-section diameter of about 2 nm as expected for an RNA strand.
However, differences were observed in finer structural details. Given
that the independently restored models yield nearly identical
scattering patterns, the ambiguity illustrated in Fig. 3a
had to be attributed to the low resolution of the scattering data.
View larger version (56K):
[in a new window]
Fig. 3.
a, low resolution models of the 5 S rRNA
obtained ab initio in five independent shape
determination runs (from left to right). The
middle and bottom rows are rotated
counterclockwise by 900 around the y and
x axes, respectively. b and c,
superposition of 10 low resolution models of the 5 S rRNA displayed as
solid and semitransparent spheres,
respectively.
Analysis of the results of independent restorations permits one to
further refine the solution and to build the most probable model of the
5 S rRNA. For this process, all of the restored configurations rotated to best match each other were remapped onto a grid of densely
packed spheres with r0 = 0.3 nm. It is
conceivable that the overlap of the 10 models in Fig. 3b
encloses the actual shape of the 5 S rRNA in solution. Already a visual
inspection of this overlap displayed as semitransparent spheres in Fig.
3c reveals the most probable shape of the particle as a
darker core formed by the dummy atoms with highest repetition rates. To
build the final model, the shape determination was performed using the
overlap in Fig. 3b (M = 705 dummy atoms) as
a search volume instead of the spherical one. The shape of the 5 S rRNA
thus obtained (Fig. 4a) is
very similar to the probable shape represented in Fig. 3b.
Independent restorations using the overlap in Fig. 3b as the search volume yielded only minor rearrangements of several dummy atom
indices on the particle border compared with the final configuration in
Fig. 4a.
|
The appearance of the ab initio model is rather similar to
that of the theoretically predicted structure of the 5 S rRNA from X. laevis by Westhof et al. (26) inasmuch as both
models consist of two elongated arms (helices). This agreement was
unexpected, as the integral parameters of the Westhof model
(Rg = 3.82 nm, Dmax = 14.0 nm) differed significantly from the experimental values, and the
calculated scattering profile (Fig. 2b, curve 4) yielded a
poor fit to the experimental curve with = 5.1. Previous models
of the free 5 S rRNA derived from SAXS data (34, 35) supported a Y
configuration with two shorter and one longer helix. In contrast, our
data indicate that the 5 S rRNA in solution consists of two longer and
one shorter helix.
The obtained shape permits one to build a tentative model of the entire
5 S rRNA using the atomic models of its fragments. The structure of the
separated and extended section including domains A, D, and E (Fig. 1)
was available from Correll et al. (29), and the rest of the
molecule (domains B and C) was taken from the Westhof theoretical model
(26). These two fragments were interactively fitted into the low
resolution model as displayed in Fig. 4a. The surface
representation of the final model of the free 5 S rRNA in solution thus
obtained is displayed in Fig. 4b.
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DISCUSSION |
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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According to the sampling theorem (37-38), the number of degrees
of freedom associated with the scattering curve in Fig. 2A is Ns =
Dmax smax/ 
20. As the
search models nominally contain M 103
parameters (indices of dummy atoms), a natural question arises whether
the information content in the scattering data justifies the ab
initio restoration. First, it should be stressed here that the
DAMs in Figs. 3 and 4a are still low resolution models, despite the
small radii of the dummy atoms used (from 0.3 to 0.4 nm). The
resolution of a DAM, and thus that of the final model in Fig. 4a, is defined solely by the range of the data fitted
(2/smax 
1.3 nm); this is best illustrated
by the models in Fig. 3a, which provide roughly the same
degree of detail despite different packing radii used. On one side, the
variety of models in Fig. 3a yielding identical scattering
curves in the fitting range demonstrates that the solution of the
ab initio shape determination problem is ambiguous. On the
other side, all of these models display similar gross structure and
differ only in the finer details. This situation resembles, to some
extent, particular problems of NMR spectroscopy, where different chain
configurations yield virtually the same discrepancy with the
experimental data. The refined shape in Fig. 4a may be
considered an analogue of the averaged structure provided by the NMR
technique. The successful alignment of the atomic models of the 5 S
rRNA fragments into this shape adds further credit to the ab
initio model.
Earlier cross-linking experiments with phenyldiglyoxal between residues 41 and 72 (39) suggested that tertiary interactions exist between domains E and C. According to our model (Fig. 4, a and b), these domains are well separated in the unbound 5 S rRNA in solution. Fig. 4c displays our model of the free 5 S rRNA along with the recent protein-RNA map of the 50 S ribosomal subunit E. coli obtained from x-ray and neutron scattering (15). The region of interest is at the top of the subunit, where an RNA fragment is complexed with three ribosomal proteins. This fragment is identified as a bound 5 S rRNA and the lower protein as L25 (15). The orientation of the free 5 S rRNA molecule in Fig. 4c was selected to have the domain E bound to L25 (40, 41). This comparison of the free and bound 5 S rRNA indicates that the 5 S rRNA becomes essentially more compact when forming a complex with specific ribosomal proteins. A conceivable structural rearrangement involves rotation of the domain B-C toward A-B-E (by about 60° clockwise in the plane of Fig. 4c). Such a rotation would also bring domains E and C closer together and enable the cross-links observed in former studies (39). Yet more information on the mechanism of the complex formation would be revealed by comparison with a bound 5 S rRNA expected to be identified in the x-ray crystallographic maps of the 50 S subunit (42).
The present ab initio model of a free 5 S rRNA provides a
basis for further crystallographic molecular replacement studies and
also for the analysis of RNA-protein complexes in solution and in crystals.
| |
FOOTNOTES |
|---|
* This research was supported by the European Union Biotechnology Program (Grant BIO4-CT97-2143 to D. I. S.) and by the Bundesministerium für Bildung und Forschung via the network of RNA Technology (RiNA) GmbH, the Deutsche Forschungsgemeinschaft (SFB 344-D6).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
49 40 89902-125; Fax: 49 40 89982-149; E-mail:
svergun@embl-hamburg.de; or Tel.: 49 40 8998-4744; Fax:
49 40 8998-4747; E-mail: betzel@unisgi1.desy.de.
Published, JBC Papers in Press, July 13, 2000, DOI 10.1074/jbc.M004974200
2 D. I. Svergun and M. H. J. Koch, unpublished results.
| |
ABBREVIATIONS |
|---|
The abbreviations used are: SAXS, small angle x-ray scattering; DAM, dummy atoms model.
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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