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INTRODUCTION |
The ribosome is the platform for all protein synthesis
and the catalyst of peptide bond formation. The centrality of the
ribosome is shown by its universality, its conservation throughout all forms of life, and its frequent targeting by toxins and antibiotics (1). The ribosome has been the subject of scores of studies that have
linked the structure of the particle with its mechanism of action in
protein synthesis. Traditional electron microscopy studies have played
a central role in allowing visualization of the ribosome and its
subunits and in the placement of component proteins, segments of its
large RNA molecules, and functional sites (2, 3).
Cryo-electron microscopy
(EM)1 adds new dimensions to
these investigations while confirming the basic observations of earlier work; it allows a description of a native particle frozen in vitreous ice and undistorted by drying and staining, and it permits
visualization of the interior of the particle rather than simply its
stained or shadowed surfaces. Cryo-EM reconstructions of the ribosome have been generated through independent investigations in two different
laboratories. This work has resulted in the attainment of greater
resolution in the description of the overall structure of the ribosome
(4, 5) and also has allowed the structural studies of ribosomes
complexed with tRNAs and protein factors (6-8). More recently, cryo-EM
data have aided in the determination of phases for crystal structures
of the 30 S subunit (9), the 50 S subunit (10), and the 70 S ribosome
(11). Crystal structures of the 30 S (12, 13) and 50 S (14) subunits
have been solved at 3.0-3.3 and 2.4 Å, respectively, and a 7.8 Å structure of a 70 S functional complex has been reported (11).
Electron density for protein L7/L12 was not apparent even in the recent
2.4 Å resolution 50 S ribosome structure (14), and the exact position
of the molecule within the ribosome remains nebulous. The protein
L7/L12 is central to the translocation step of translation, and it is
the only ribosomal protein that is present in multiple copies (for
review see Ref. 15). The monomer of L7/L12 has a molecular mass of 12 kDa and is organized in the ribosome as two dimers bound to one copy of
protein L10, which in turn anchors the pentamer to the large subunit.
The L7/L12 monomer includes two distinct structural domains that are
thought to be linked by a flexible hinge (residues 37-52); the
elongated helical N-terminal domains (residues 1-36) are responsible
for dimer interaction and binding to L10, and the larger globular C-terminal domains (residues 53-120) interact with elongation factors.
The crystal structure of the Escherichia coli L7/L12 C-terminal domain has been solved at 1.7 Å resolution (16). Models of
the E. coli L7/L12 indicate that the dimer can span up to
125 Å when extended (17). A recent crystal structure of L12 from the
hyperthermophilic bacterium Thermotoga maritima shows two alternative conformations for the hinge region, an extended coil or
a long
-helix that folds back on the N-terminal domain forming a
compact overall protein structure (18). The flexible hinge conformation
agrees better with the body of evidence that indicates that the
E. coli L7/L12 protein has a great deal of overall
flexibility (19).
L7/L12 is easily and selectively dissociated from and reconstituted
into the ribosome (20). When it is removed, one of the most
recognizable features seen in images of negatively stained 50 S
particles such as the stalk (or arm) disappears. The stalk and full
activity are restored when L7/L12 is added back, and the ribosome
nomenclature has equated L7/L12 and the stalk. A body of evidence
suggests that L7/L12 exists in at least two conformations, and its
mobility is essential for its function (19, 21, 22).
The experiments described in this study demonstrate a direct approach
to the placement of ribosomal proteins that is based on ribosome
reconstitution and is related to the classic "single omission
reconstitutions" of Nomura (23). The cryo-EM structure of 70 S
ribosomes, which lack a single protein component, L7/L12, is compared
with that of the reconstitutes, which include the protein. Some
conformational changes are observed in the ribosome upon the binding of
L7/L12 in regions previously noted to be conformationally flexible (4,
5). In addition, we have incorporated a variant form of L7/L12 that has
been modified with Nanogold, a 14 Å gold complex (24) that is easily
seen in cryo-electron micrographs. Difference mapping, together with
Nanogold labeling, places the N-terminal domains of protein L7/L12 on
the 50 S subunit body next to protein L11 and indicates four allowed
sites for the L7/L12 C-terminal domains.
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EXPERIMENTAL PROCEDURES |
Preparation and Characterization of Ribosomes--
E.
coli ribosomes were isolated (25), and tight couples were prepared
as described previously (26). The treatment of 70 S ribosomes with 0.5 M NH4Cl and 50% ethanol at 0 °C dissociated protein L7/L12 and generated cores that were isolated by sedimentation (27). Recombinant wild type and Cys-89 L7/L12 were prepared as
described previously (28). Incubation of 70 S core particles with an
8-fold molar excess of either native or Nanogold-modified L7/L12
yielded wild type and Nanogold-modified reconstituted ribosomes, respectively. Wild-type reconstitutes were separated from free protein
by sedimentation through a 10% sucrose pad (28). Nanogold-modified reconstituted 70 S ribosomes were isolated on 10-30% sucrose
gradients in 10 mM Tris-HCl, pH 7.5, 60 mM
NH4Cl, and 10 mM MgCl2 by
centrifugation in a Sorvall AH-650 rotor at 40,000 rpm for 3 h at
4 °C. The fractions, which contained 70 S ribosomes, were used for
microscopy after dialysis against 10 mM Tris-HCl, pH 7.5, 60 mM NH4Cl, and 10 mM MgCl2. Ribosomes and reconstituted ribosome preparations
were analyzed by electron microscopy of negatively stained samples as
described previously (25). To observe 50 S subunits, portions of our
ribosome preparations were dissociated by incubation in buffer
containing 1 mM Mg2+ and were negatively
stained. The methods used in the extraction of ribosomal proteins (29)
and their analysis by reverse-phase HPLC (30) and mass spectrometry
(31) were adapted from these previously published procedures.
Preparation of Nanogold-labeled Protein L7/L12--
Immediately
after reduction with 1% 2-mercaptoethanol in 10 mM
Tris-HCl, pH 7.5, and 200 mM NH4Cl for 1 h
at 37 °C and the removal of the reducing agent by passage through
two successive Bio-Rad Bio-Spin 6 columns equilibrated in 20 mM sodium phosphate, pH 6.5, approximately 4 nmol of
recombinant Cys-89 L7/L12 in 70-80 µl of 20 mM sodium
phosphate, pH 6.5, were added to an excess of monomaleimido-Nanogold
reagent (Nanoprobes Inc., Stony Brook, NY) suspended in 30 µl of
isopropyl alcohol. Water was added to obtain a final volume of
180 µl, and the reaction mixture was incubated for 1 h at room
temperature. Nanogold-modified protein was isolated by size exclusion
HPLC on a Beckman Spherogel-TSK 2000 SW column (7.5 × 300 mm) at
a flow rate of 1 ml/min. 20 µl of aliquots of each of the 500-µl
fractions were run on duplicate 12.5% Laemmli SDS-polyacrylamide gels
(32). One gel was stained for proteins with silver stain (Bio-Rad), and
the other gel was stained for Nanogold with the LI Silver stain
(Nanoprobes, Inc.). The fractions with the highest concentration of the
separated labeled protein were concentrated and used for ribosome
reconstitution as described above.
Cryo-electron Microscopy--
Ribosome samples were prepared for
cryo-electron microscopy according to previously published procedures
(33, 34). A 5-µl droplet of sample was applied to a holey
carbon-coated molybdenum grid that was freshly coated with a thin
(~20 Å) carbon film. After 30-50 s, the grid was blotted
from both sides for 2-3 s (35) and plunged into an ethane slush. The
grid was immediately transferred to and stored in liquid nitrogen.
Microscopy was performed on a Philips CM120 transmission cryo-electron
microscope (FEI, Hillsboro, OR) equipped with a LaB6
filament, a Gatan cryo-holder (Gatan, Pleasanton, CA), and a Gatan
slow-scan charge-coupled device (CCD) camera (yttrium-aluminum
garnet (YAG) scintillator, 1024 × 1024 pixels). Images were
collected under low dose conditions (<20 electrons/Å2) at
a nominal magnification of × 45,000 and at three defocus levels
(
0.5,
1.0, and
1.5 µm). The digital images have a pixel size of
4.1 Å as determined by calibration with a catalase crystal.
Image Processing--
Interactive selection of individual
ribosome images as 100 × 100 pixel fields, the exclusion of
density from nearby particles, and the application of a circular mask
were performed with the QVIEW software package (36). Subsequent
processing was performed with the IMAGIC software package (37).
Initially, a set of 4447 images of 70 S tight couple particles
(collected at
1.5 µm defocus and low-pass filtered to 30 Å and
density inverted) was translationally aligned and then subjected to
multivariate statistical analysis and classification essentially as
described previously (38) to obtain 500 class-sum images. The
Euler angles of the 347 class-sum images with the highest membership
were calculated by angular reconstitution, and a preliminary
three-dimensional reconstruction was calculated by exact filtered back
projection (39). After three rounds of refinement by the anchor set
method (37), the three-dimensional reconstruction of the 347 class-sum
images was used as an initial reference to find the Euler angles of the
individual 4447 particle images, low-pass filtered to 23 Å. After two
rounds of refinement, the final reconstruction of these images was used in turn as an initial reference model for the determination of the
Euler angles of images of L7/L12-reconstituted ribosomes and core particles.
Images collected at three different defocus values (
0.5,
1.0, and
1.5 µm) were combined analytically after deconvolution and
correction for the contrast transfer function of the electron microscope as described previously (38), with the exception that the
decay constant was 25 nm2, and the Fermi filter resolution
cutoff was 1/8.2 Å
1. The data sets of
L7/L12-reconstituted ribosomes and core particles each underwent three
rounds of anchor set refinement. The final reconstruction of
L7/L12-reconstituted ribosomes included data from 8565 images, and that
of the core particles included data from 9739 images. The Euler angles
for 486 contrast transfer function-corrected particle images of
Nanogold-labeled ribosomes were determined by using the wild type
L7/L12-reconstituted ribosome reconstruction as a reference. After
calculating a preliminary Nanogold-labeled reconstruction, a subset of
the best 317 Nanogold-labeled particle images was selected by
comparison of the particle images with their corresponding reprojections.
The resolution of the reconstructions was calculated by the Fourier
shell correlation method, splitting the data sets into two halves,
calculating two half-reconstructions, and assuming the 0.5 correlation
threshold as the resolution limit (40). Ribosome reconstructions were
displayed with the AVS visualization package (Advanced Visualization
System, Inc.) at isosurface threshold levels chosen to include a volume
of 2.9 × 106 Å3. The difference map
between the wild type L7/L12-reconstituted ribosome and the core
ribosome was calculated in IMAGIC after filtering the input maps to a
26 Å resolution. Difference maps were also calculated using
reconstructions obtained from randomly generated half-sets of our core
and reconstituted ribosome images. These maps gave the same results as
the one with the entire data sets. Difference density not clearly
associated with the ribosome was removed with a circular mask (260 Å diameter), and small regions of disconnected density were
computationally removed. The difference density between the
reconstituted and core ribosome reconstructions is displayed with the
isocontour level set at three standard deviations (3
) above the mean value.
The resolution of the Nanogold-labeled reconstruction was evaluated
both by the Fourier shell correlation between two
half-reconstructions and by the Fourier shell correlation between the
full non-split Nanogold reconstruction with the full reconstruction of
the wild type L7/L12-reconstituted ribosome. Difference maps were
calculated between the Nanogold-labeled ribosome and the wild type
reconstituted ribosome after filtering the input maps to either 45 Å or 60 Å resolution. The 45 Å resolution Nanogold difference density
is displayed with the isocontour level set at five standard deviations (5
) above the mean value.
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RESULTS |
Preparation and Characterization of Cores and Reconstituted
Ribosomes--
E. coli ribosomes and tight couples were
prepared by well established procedures (25). L7/L12 is easily
extracted from ribosomes as described previously (for details see Refs.
20 and 22). We chose the gentlest possible condition that afforded the
maximal removal of L7/L12 and the minimal extraction of other ribosomal proteins. 70 S tight couples were treated with NH4Cl and
ethanol on ice, and the resulting 70 S ribosome cores were isolated by sedimentation. It is well established that L7/L12 forms the
characteristic stalk observed in negatively stained 50 S subunits (41).
To confirm the removal of L7/L12 in our core preparation, we used EM to
examine negatively stained 50 S subunits of our ribosome preparations.
In the tight couple 50 S subunits, the L7/L12 stalk was readily
identified, whereas in the 50 S subunits of the cores, a complete
absence of stalks was noted. To establish that recombinant wild
type L7/L12 or Nanogold-labeled L7/L12 is incorporated specifically into ribosomes, we viewed 50 S subunits of the reconstitutes and observed nearly complete restoration of stalks.
The protein composition of our ribosome preparations was confirmed by
HPLC and electrospray ionization mass spectrometry. Ribosomal proteins
were extracted from 70 S tight couples, 70 S cores, and L7/L12 70 S
reconstituted ribosomes and analyzed by reverse-phase HPLC. In Fig.
1A, the arrow
identifies a double peak that elutes last in the chromatogram of the
tight couple ribosomal protein extract. This double peak elutes at the
same position as a purified L7/L12 standard and contains a species of
molecular mass compatible with protein L7/L12 as determined by mass
spectrometry. The elution profile of an extract from 70 S cores lacks
the L7/L12 double peak (Fig. 1B), whereas the L7/L12 double
peak is restored in the chromatogram of the protein extract from
L7/L12-reconstituted ribosomes as shown in Fig. 1C. No other major differences were observed among the chromatograms. Also, the
analysis of the supernatant of the NH4Cl/ethanol extract of tight couples revealed that the protein L7/L12 accounted for
approximately 95% of the protein, and that none of the minor peaks was
compatible with ribosomal proteins. We conclude that our means
of extraction of L7/L12 yields cores that are greatly depleted in
protein L7/L12 while not removing any significant amounts of other
ribosomal proteins. In addition, the cores incorporate purified wild
type or Nanogold-labeled L7/L12 protein efficiently. The level of
L7/L12 in the reconstituted ribosomes was estimated to be ~75% by
evaluating the relative peak areas in the HPLC profiles and >90% by
EM of negatively stained samples.

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Fig. 1.
HPLC of ribosomal protein extracts.
A, extract from 300 pmol of 70 S ribosome tight couples.
B, extract from 300 pmol of 70 S cores. C,
extract from 25 pmol of reconstituted ribosomes.
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Structures of Wild Type L7/L12-reconstituted Ribosomes and of 70 S
Core Particles and Difference Mapping--
Our goal was to separately
determine and then compare the structures of ribosomal core particles
and reconstitutes as a means of identifying the location of protein
L7/L12 within the ribosome. A preliminary reconstruction of 70 S tight
couples was used as a reference model for determining the orientational
angles of particle images of both 70 S cores and L7/L12-reconstituted
ribosomes. After correction of the particle images for the contrast
transfer function of the electron microscope and three rounds of
refinement, the two final reconstructions had a resolution of 26 Å at
the 0.5 correlation cutoff of the Fourier shell correlation (Fig. 2) (40). Three-dimensional maps of the
L7/L12-reconstituted ribosome (green) and the 70 S cores
(blue) are shown in Fig. 3. As
shown by HPLC and mass spectroscopy, the preparations of
L7/L12-reconstituted ribosomes and the 70 S cores differ only in the
four copies of protein L7/L12. The absence of this protein results in a
difference of 48 kDa in the 2.5-MDa mass of the entire ribosome. Thus,
difference mapping is needed to visualize the location of L7/L12. A
difference map was calculated between the reconstituted and core
particle reconstructions revealing significant difference density in
the L7/L12 shoulder region (red). The volume of the observed
difference density corresponds to 19 kDa of protein. Because the
N-terminal domain is known to anchor L7/L12 to the ribosome and the
hinge region is flexible, we propose that the difference density
corresponds to four copies of the N-terminal domain (15 kDa) and a
portion of the four hinge regions (4 of 6 kDa). We presume that the
C-terminal domains of L7/L12 are not observed in the difference map
because of either static or dynamic structural disorder.

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Fig. 2.
Resolution assessment of cryo-EM
reconstructions. Fourier shell correlation functions between two
independent reconstructions of L7/L12-reconstituted ribosomes
(dark solid line) and 70 S cores (gray
solid line) shown with the 3 -threshold curve (broken
line).
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Fig. 3.
Two ribosome reconstructions and the
difference map filtered to 26 Å resolution.
Green, L7/L12-reconstituted ribosomes;
blue, 70 S cores; red, difference map between
reconstituted ribosome and core reconstructions. The L1 and L11 regions
as well as the central protuberance (CP) are labeled on the
reconstituted ribosome reconstruction. A, a view toward the
50 S side. B, a rotated view to correspond to a specific
orientation of the 50 S subunit (for details see Fig. 7
(bottom) of Ref. 5). The two arrows denote a
protein and a rRNA fragment modeled by Gabashvili et al.
(5). C, a view from the top with the 30 S on the
left.
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Small conformational changes are observed throughout the ribosome,
specifically in the regions assigned to L1, L9, the 30 S head, and the
base of the 50 S subunit. The L1 region of the core structure is
smaller and less well defined than in the reconstituted structure,
consistent with the observation of local structural flexibility in the
L1 protein region (4, 42). Differences are also observed below the L1
region where protein L9 has been localized by immune electron
microscopy (43). The structure of protein L9 has been determined by a
combined use of x-ray crystallography and NMR (44). Matadeen
et al. (4) have fit the atomic structure of L9 within their
cryo-EM reconstruction of the 50 S subunit, and they propose a shift of
~50 Å for this protein in the intact ribosome. We also note
conformational changes in the region of the 30 S head and neck, which
are not surprising because Agrawal et al. (42) have found
that the 30 S neck is one of the most flexible regions of the ribosome.
The 30 S head appears to be held closer to the central protuberance of
the 50 S subunit in the reconstituted ribosome structure.
Our position for the N-terminal region of L7/L12 found by difference
mapping (Fig. 3) is compatible with the extensive literature that
localizes the protein to this region of the ribosome (17). Specifically, we find L7/L12 next to the inferred location of L11. The
crystal structure of an L11·RNA complex (45) was positioned into the 5 Å resolution crystallographic density map of the
Haloarcula marismortui 50 S subunit (46). The crystal
structure of the L11·RNA complex (45) was also fit in the 11.5 Å resolution cryo-EM density map of the E. coli 70 S ribosome
(5). Because it is easier to compare two cryo-EM structures, we include
our difference map in one orientation (Fig. 3B) matching
that of a figure in Gabashvili et al. (5). In this view, the
elongated density assigned to L11 is observed to fold over the L7/L12
difference density. Our finding of neighboring locations for L7/L12 and
L11 is consistent with previous cross-linking experiments that show both the N-terminal and C-terminal domains of L7/L12 are adjacent to
L11 (17).
Cryo-electron Microscopy of Nanogold-labeled L7/L12-reconstituted
Ribosomes--
The analysis of ribosomes reconstituted with
Nanogold-labeled L7/L12 allowed the placement of the C-terminal
domains. Because native L7/L12 lacks cysteine, a mutant form Cys-89
L7/L12 (28) was used. Derivatization of the single cysteine with
Nanogold produced protein that was labeled near the tip of the
C-terminal domain as shown in Fig. 4.
After the Nanogold reaction, the protein was isolated from any
unreacted reagent by HPLC, and two SDS-polyacrylamide gels were run;
one stained for protein, and the other stained for Nanogold. The extent
of Nanogold modification of L7/L12 was judged to be nearly 100%
immediately after the isolation of the labeled protein. After storage
of the labeled protein for 1 week, gel analysis indicated that only a
negligible percentage (<5%) of the L7/L12 still contained Nanogold.
Thus, the subsequent steps involved in the preparation of reconstituted
ribosomes with Nanogold-labeled L7/L12 and purification of 70 S
ribosomes by sucrose gradient centrifugation and dialysis were carried
out as quickly as possible. Cryo-EM grids were prepared on the second
day after isolation of the Nanogold-labeled L7/L12 protein. Cryo-EM
images of the Nanogold-labeled L7/L12-reconstituted ribosomes indicated
that only ~30% of the particles contained Nanogold. Because negative stain EM analysis indicated >90% incorporation of Nanogold-labeled L7/L12 as was found for the wild type L7/L12 reconstitution, we conclude that a significant loss of the gold label occurred during the
preparation and isolation of the Nanogold-labeled 70 S ribosomes. Another compounding factor that led to the collection of a much smaller
cryo-EM data set for the Nanogold-labeled ribosomes than for either of
the reconstituted wild type or core ribosomes was that the
Nanogold-labeled sample was ~10 times less concentrated. Thus, for
the Nanogold-labeled sample fewer ribosomes were observed in each
digitally collected cryo-electron micrograph (1024 × 1024 pixels).

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Fig. 4.
Structure of ribosomal protein L7/L12 and a
Nanogold derivative. A, a diagram of the
domain structure of L7/L12. B, a crystal structure of the
C-terminal domains of the L7/L12 dimer showing the sites of the Cys-89
mutation. C, a schematic drawing of the
maleimido-Nanogold complex bound to the protein. D, a
diagram of L7/L12 labeled with Nanogold.
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Fig. 5A shows examples of
cryo-EM particle images preprocessed in QVIEW (36) of Nanogold-labeled
ribosomes. Only particles that clearly showed gold clusters, visible as
intense dark spots, were selected leading to a total data set of 486 particle images. These particle images contained 1-4 gold clusters
each. Even if there were no loss of the gold label, four well separated
gold clusters would not have been seen necessarily in each particle image because of the particle orientation. Thus, no attempt was made to
select for particle images of ribosomes with all four possible sites
labeled. Orientational angles were determined for the Nanogold-labeled
ribosome particle images using the reconstruction of the wild type
L7/L12-reconstituted ribosome as a reference. A preliminary
reconstruction was calculated from the full Nanogold-labeled ribosome
data set. A comparison of the Nanogold-labeled ribosome particle images
with projections of the preliminary reconstruction indicated that
correct orientational angles were only found for ~65% of the
particle images. Given the high probability that some gold label was
lost and not all of the ribosomes in the data set had the same
complement of sites labeled, we chose not to refine the incorrect
orientational angles but rather to select the subset of particle images
with apparently correct orientational angles. Thus, a relatively small
set of 317 Nanogold-labeled ribosome particle images was used to
generate the final Nanogold-labeled ribosome reconstruction. The
resolution of the Nanogold-labeled reconstruction was assessed in two
ways, first by calculating a Fourier shell correlation plot between two
half-reconstructions and second by calculating a Fourier shell
correlation plot between the reconstruction of the full Nanogold data
set with the reconstruction of the wild type reconstituted ribosome.
Given the small size of the data set, it is not surprising that
splitting the data set gave a poorer resolution of 60 Å than compared
with the wild type reconstruction, which indicated a 45 Å resolution.
The better resolution estimate seems more correct because filtering the
Nanogold reconstruction to 60 Å excessively smoothes real
structural features that can be observed in the Nanogold reconstruction
filtered to a 45 Å resolution (Fig. 5, B-E).

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Fig. 5.
Cryo-electron microscopy of Nanogold-labeled
L7/L12-reconstituted ribosomes. A, raw images
preprocessed in QVIEW of Nanogold-labeled ribosomes taken at 1.5 µm
defocus. B-E, reconstruction of the Nanogold-labeled
reconstituted ribosome filtered to 45 Å resolution. B, a
view toward the 50 S side. C, a side view with the 30 S on
the left. D, a view toward the 30 S side.
E, a top view with the 30 S on the left.
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Localization of L7/L12 by Nanogold Labeling--
The sites of
Nanogold density were visualized by subtracting the structure of the
wild type L7/L12-reconstituted ribosome from the Nanogold-labeled
L7/L12 ribosome reconstruction after filtering both density maps to 45 Å resolution. The difference mapping was repeated with both input
density maps filtered to a 60 Å resolution, and the same Nanogold
sites were observed. The Nanogold difference map revealed four clusters
of density (yellow) shown together with the density
attributed to the N-terminal region of L7/L12 (red) and the
core particle reconstruction (blue) in Fig.
6. One of the four Nanogold sites,
density II, is adjacent to the L7/L12 density (red), whereas
the centers of Nanogold sites I, III, and IV are each about 80-90 Å away. This distance is consistent with the length of the
maleimido-Nanogold label (20 Å) plus the length of the C-terminal
domain of L7/L12 (35 Å) and a portion of the hinge region (50 Å maximum).

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Fig. 6.
Localization of L7/L12 within the 70 S
ribosome. Yellow, Nanogold density (isocontoured at
5 ) from the difference map between the Nanogold-labeled
L7/L12-reconstituted ribosome and the wild type L7/L12-reconstituted
ribosome reconstructions (both filtered to 45 Å resolution);
red, density difference attributed to L7/L12 from Fig. 3;
blue, 70 S cores from Fig. 3. In the left panels,
the 70 S core provides a reference for the locations of the difference
densities within the ribosome. In the right panels, the core
is not shown so that the full extent of the L7/L12 density
(red) and the four gold density regions are visible.
A, a side view with the 30 S on the left and the
50 S on the right. B, a top view as in Fig.
3C with the 30 S on the left.
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The largest density attributed to Nanogold (labeled I) localizes on the
30 S subunit head on the side facing the 50 S subunit. The second
largest density (labeled II) is elongated and is situated on the body
of the 50 S subunit below the L7/L12 difference density (red). An additional Nanogold density is present just
below the 50 S central protuberance on the side of the L1 shoulder
where it is present as two small masses (labeled III) and high on the body of the 30 S subunit (labeled IV). Our interpretation of these observations with respect to the location of protein L7/L12 is summarized in the models presented in Fig.
7.

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Fig. 7.
Models of allowed conformations of
L7/L12 within the 70 S ribosome. All of the panels show
the same conformation for one L7/L12 dimer with the C termini at the
presumed high affinity site on the 50 S subunit corresponding to the
Nanogold label site II in Fig. 6. Three conformations are shown for the
C termini of the second L7/L12 dimer corresponding to the Nanogold
label sites I (A), III (B), and IV
(C). The ribosome is oriented with the 30 S on the
left and the 50 S on the right.
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DISCUSSION |
Location of Protein L7/L12 within the E. coli 70 S
Ribosome--
The difference map between core particles and
reconstituted ribosomes has allowed us to visualize the location of
L7/L12 N-terminal domains, which interact strongly with the 50 S
subunit, whereas the intense density of the Nanogold label lets us mark
the positions of the mobile C-terminal domains. As illustrated in Fig.
7, we postulate that the flexible L7/L12 hinge enables the paired
C-terminal domains to localize to at least four discrete positions in
the 70 S ribosome but not to all four sites simultaneously. The
electron dense Nanogold label has allowed us to capture these partially occupied sites. The evidence for a different conformation, location, and function of each dimer is plentiful (22), and each site identified
in our work is compatible with the biochemical data.
In all the panels of Fig. 7, one L7/L12 dimer is shown in a
conformation in which the hinge bends and contracts to bring the C-terminal domains onto the 50 S body adjacent to the N-terminal domains. The hinge of this dimer could be in a rigid arrangement and
may account for the narrow tail of difference density that extends
toward Nanogold site II (Fig. 6). We postulate that this conformation
corresponds to the high affinity site first described by Zantema
et al. (47). The domain placement is consistent with localizations by immune electron microscopy (41), with cross-linking (e.g. to proteins L10 and L11) (22), and with energy
transfer studies with native and mutant proteins (21). Fig. 7,
A-C, shows conformations of the second L7/L12 dimer in
which the hinge extends into the interface cavity, allowing the
C-terminal domains to reach sites that are distant from the
"so-called" L7/L12 shoulder. The site on the 30 S head (Fig.
7A) is compatible with the cross-linking, which is enhanced
by the elongation factor Tu, to 30 S subunit proteins S7 and S14
(22). Fig. 7B shows the C-terminal domains located
below the central protuberance in proximity to the peptidyltransferase center and at a location compatible with the observed cross-links to
the 50 S subunit proteins L2 and L5 (22, 48, 49). Finally, Fig.
7C shows a conformation in which the protein extends
straight toward the head and/or body junction of the 30 S subunit, and the C-terminal domains are at a location compatible with the
cross-links to S2 and S3 (50).
Given the flexibility and extended nature of L7/L12, these sites of the
L7/L12 C-terminal domains are all within reach of the difference
density we attribute to the N-terminal anchors. The variation observed
in the size of the four Nanogold density sites may relate to the fact
that within a single dimer the relative separation and orientation of
the C-terminal domains have been shown to vary (21, 51). In our
interpretation, the four sites identified by Nanogold labeling most
probably correspond to alternative L7/L12 conformations and illustrate
the mobility of the C-terminal domains of protein L7/L12 within the ribosome.
Long Range Interactions and Conformational Changes in the
Ribosome--
In comparing our reconstructions of the reconstituted
and core ribosomes, we noted some conformational changes in the L1 and L9 region on the opposite side of the ribosome from the L7/L12 shoulder. This is consistent with evidence for a long range interaction between the two sides of the ribosome, e.g. the binding of
an antibody to protein L9 below the L1 shoulder strongly interferes in
the translocation factor binding and function that occur in the L7/L12
region (52).
Conformational changes upon the subunit association and the binding of
factors have been reported for cryo-EM (4, 6, 8, 42, 53, 54) and x-ray
(11, 46, 55) structures. In addition, conformational changes associated
with tRNA (6, 7, 56) and mRNA binding (57) and translocation (53)
are well documented. The ribosome also alters its conformation in response to changes in divalent cations (58) and during its ordered
assembly from RNA and proteins (23). The incorporation of L7/L12 into
core particles also appears to cause conformational changes throughout
the ribosome as shown by our results.
It has been assumed that all four copies of protein L7/L12 are present
in the 50 S stalk because electron micrographs of negatively stained 50 S particles that lack L7/L12 show no stalk, and the reincorporation of
the protein restores this structure. This generalization, although
challenged as early as 1982 by Zantema et al. (47), remains
prevalent in the literature. Monoclonal antibody-labeling studies on
isolated 50 S subunits have shown that the C-terminal region of L7/L12
can be localized both to the end of an extended stalk and to a site on
the body of the 50 S subunit (41). Our Nanogold-labeling study
indicates four sites for the C-terminal domain of L7/L12 in the
reconstituted 70 S ribosome, two of which are located on the 30 S
subunit. An extended stalk has only been observed in cryo-EM
reconstructions of the ribosome complexed with other molecules, such as
tRNAs and elongation factor G (40, 53). The absence of an extended
stalk in other cryo-EM reconstructions has been explained by the
presumed flexibility of the stalk and its ability to fold back onto the
body of the 50 S subunit (6, 10, 59). Our difference mapping and
Nanogold-labeling studies indicate that the two dimers of L7/L12 are
able to fold back onto both the 30 S and the 50 S subunits of the 70 S ribosome.
Functional Implications of the Location of L7/L12--
Our results
show the hinge regions and C-terminal domains of at least one dimer of
protein L7/L12 to be present in the intersubunit cavity in an area
occupied by the elongation factors upon interaction with the ribosome.
Elongation factor G has been placed in this part of the ribosome by
difference mapping of cryo-EM reconstructions (53, 60). The factor
interacts with the 50 S subunit in the area we associate with the
N-terminal domains and the C-terminal domains of the high affinity site
(Fig. 6, density II). An additional contact of elongation factor G is
to the 30 S subunit at the subunit neck and not far from densities III
and IV of Fig. 6. The placement of tRNA molecules in cryo-EM (7, 56)
and x-ray (11) structures shows contact regions on the small subunit in
the region of densities I and IV, and density III is very near the
peptidyltransferase center of the large subunit. Cross-linking data
show that the binding of an elongation factor Tu·tRNA complex alters
the frequency of formation of specific links, e.g.
cross-linking from Cys-89 to L5 (near density III), L10 and L11 (near
density II), and S3 (beside density IV) are decreased, whereas the
links to S7, S14, and S18 (in the 30 S head and platform) are increased
(22). This observation suggests the movement of the C-terminal domains through the cycle of the addition of each amino acid and raises the
possibility that additional stable sites for this element of the
protein exist at different stages of the translation cycle.
In conclusion, we have localized the N-terminal domains of protein
L7/L12 within the E. coli 70 S ribosome on the body of the
large ribosomal subunit and used Nanogold labeling to identify allowed
sites for the C-terminal domains. Our results provide further evidence
for the mobility of the C-terminal domains of the protein within the
ribosome, as well as for the conformational flexibility of the ribosome itself.