Molecular Cell, Vol. 8, 181–188, July, 2001, Copyright 2001 by Cell Press
The Polypeptide Tunnel System in the Ribosome
and Its Gating in Erythromycin Resistance Mutants
of L4 and L22
The bacterial ribosome is the target for many antibiot-
ics, which act by interfering with steps in the translation
cycle, for example, factor binding, tRNA binding, pep-
(Spahn and Prescott, 1996). Macrolides are a group of
antibiotics that act in the vicinity of the peptidyltransfer-
ase center and the entrance of the polypeptide tunnel.
Among these, a subgroup of smaller macrolides that
block the tunnel (Arevalo et al., 1988). In line with this
hypothesis and recent X-ray coordinates of the ribo-
somal 50S subunit, minor modifications in the terminal
regions of the tunnel-lining proteins L4 and L22 produce
resistance to these drugs (Brisson-Noel et al., 1988;
Mazzei et al., 1993; Spahn and Prescott, 1996). The
mutations work in two different ways, either by pre-
venting the drug binding (L4) or by neutralizing the ef-
fects of the binding (L22) (Wittmann et al., 1973; Pardo
and Rosset, 1977). Therefore, structural differences be-
tween the two mutants can be expected to give insights
into the mechanism of binding and mode of inhibition
by erythomycin. In addition, a comparison of mutant
provide information on the mode of drug action and,
possibly, the mechanism of peptide translocation.
We undertook a cryo-electron microscopy (cryo-EM)
study of E. coli 70S ribosomes with L4 and L22 proteins
containing erythromycin resistance mutations. In the
course of the study, a detailed analysis of the topology
of the main tunnel, considered to be the conduit for the
nascent polypeptide, revealed the existence of an entire
branched system of tunnels communicating with the
solvent through four exits. While the purpose of the
that multiple routes might exist that are dynamically
controlled and that both L4 and L22 have a pivotal role
in such a regulation.
A number of structural changes were observed, the
most significant being a substantial narrowing of the
tunnel entrance in the L4 mutant, which does not bind
erythomycin, making the opening slightly smaller than
an erythromycin A molecule. In contrast, the L22 mutant
ribosome, which binds erythromycin but is not inhibited
by it, had an opening at least twice that size. This obser-
vation supports a simple steric-hindrance mechanism
for the action of this drug by binding inside the tunnel
and blocking it, thus preventing the growth of the poly-
peptide chain. A comparison with cryo-EM reconstruc-
tions of E. coli 70S ribosomes in unbound and various
tRNA- and EF-G-bound states suggests that opening
and closing of the tunnel entrance might be an inherent
dynamic feature of the ribosome, which could, in part,
be regulated by proteins L4 and L22. These structural
rearrangements in the tunnel entrance are correlated
with conformational changes in the whole interface can-
yon, including regions responsible for binding with the
acceptor ends of tRNAs and the 30S subunit in the L7/
L12 stalk region and the central protuberance.
Irene S. Gabashvili,1Steven T. Gregory,4Mikel Valle,1,2
Robert Grassucci,1,2Michael Worbs,5
Markus C. Wahl,5Albert E. Dahlberg,4
and Joachim Frank1,2,3,6
2Howard Hughes Medical Institute
3Department of Biomedical Sciences
State University of New York at Albany
P.O. Box 509
Albany, New York 12201
4Department of Molecular and Cell Biology
Division of Biology and Medicine
Providence, Rhode Island 02912
5Max-Planck-Institut fu ¨r Biochemie
Am Klopferspitz 18a
Variations in the inner ribosomal landscape determin-
ing the topology of nascent protein transport have
been studied by three-dimensional cryo-electron mi-
croscopy of erythromycin-resistant Escherichia coli
70S ribosomes. Significant differences in the mouth of
study support a simple steric-hindrance explanation
for the action of the drug. Examination of ribosomes
in different functional states suggests that opening
and closing of the main tunnel are dynamic features of
the L7/L12 stalk region. The existence and dynamic
behavior of side tunnels suggest that ribosomal pro-
teins L4 and L22 might be involved in the regulation
of a multiple exit system facilitating cotranslational
processing (or folding or directing) of nascent pro-
The ribosome is a multifunctional machine that trans-
lates the message encoded in mRNA, builds a polypep-
tide according to this program, and provides facilities
for initial folding of the preprotein and its delivery to the
final destination. The nascent polypeptide chain exits
1982; Milligan and Unwin, 1986; Yonath et al., 1987,
Frank et al., 1995) that traverses the large ribosomal
subunit and is paved mainly by RNA loops (Ban et al.,
2000). Its middle segment, however, is fenced by long
extensions of proteins L4 and L22, reaching the tunnel
from the solvent side of the ribosome (Ban et al., 2000;
Nissen et al., 2000).
Figure 1. Three-Dimensional Cryo-EM Maps of E. coli Ribosomes Resistant to Erythromycin and Wild-Type Control
(A, D, and G) Mutation of L4. (B, E, and H) Mutation of L22.
(C, F, and I) Wild-type control (Gabashvili et al., 2000). (A–C) Solvent-side view of the 70S particles. (D–F) Interface view of the 50S subunit
portion of the maps. (G–I) Close-ups of the region around the tunnel entrance, the view shown in (D–F). A molecule of erythromycin, oriented
to show its bigger dimensions, is also shown for rough size comparison. In (G), the positions of helices h38 and h69 of 23S rRNA, constituting
bridges B1a and B2a, are marked. (Note that the zoomed portion in [G–I] does not show the complete opening, as part of it is hidden behind
an elevation next to h69). Features marked are as follows: St, L7/L12 stalk; L1, L4, and L22, globular portions of proteins of the large subunit;
sp, spur; e1, e2, e3, and e4, exits of the tunnel system; e1 is the primary “polypeptide translocation” exit. Scale bar ? 30 A˚.
et al., 1999; Gabashvili et al., 2000) further away from
the 50S subunit body. Another remarkable distinction
between the maps is at the L7/L12 stalk, which appears
extended and well defined in the case of the L22 mutant
(Figure 1e), but apparently destabilized and positionally
undefined in the case of the L4 mutant (Figure 1d). The
head of the 30S subunit undergoes a slight counter-
clockwise rotation (in the direction from A to P site),
sons of the cryo-EM density maps of the mutant ribo-
somes with 15 reconstructions previously obtained in
vili et al., 1999, 2000; Malhotra et al., 1998; Spahn et al.,
1999; Agrawal et al., 2001) all show significant variations
Long-Range Structural Effects of the Mutations
The structures of erythromycin-resistant ribosomes ob-
tained by cryo-EM and image reconstruction tech-
niques, both at ?17 A˚resolution, are shown in Figure
1, along with a higher resolution initiator tRNA-bound
ribosome (Figures 1a–1c, 70S particles; Figures 1d–1f,
50S-subunit portion of the maps, with tRNA masked off
in Figure 1f). A set of large-scale distortions can be
easily noticed in the morphology of the mutants. The
main differencesare locatedin theregions ofthe central
protuberance and the interface canyon. Particularly, the
L22 mutation results in a raised position for the 5S RNA
(CP in Figure 1) and moves bridge B2a (Figure 1; Cate
Gating Mechanisms of the Polypeptide Tunnel System
around the mouth of the tunnel. The changes affect the
appearance of helix 69 of 23S RNA that is known to
form a bridge (B2a) linking the 50S and 30S subunits
(Cate et al., 1999; Gabashvili et al., 2000). Surface fea-
tures of the L22 mutant are closer to those of the wild-
type ribosomes complexed with EF-G in its GDP state
(Agrawal et al., 1998, 1999), although there is dissimilar-
ity among the maps in the A-site region. Overall, the
structure of the ribosome with the mutated L4 protein
resembles that of the empty ribosome, although there
are some variations between the 30S subunit portions
of the maps. A ram mutation in helix 27 of the 30S
subunit (Lodmell and Dahlberg, 1997; Gabashvili et al.,
helix 27 mutant exhibits larger differences in the inter-
mutations in helix 69 (part of B2a) also cause a ram
phenotype (O’Connor and Dahlberg, 1995).
The most interesting observation is that the ability of
the ribosome to bind erythromycin is correlated with the
width of thetunnel opening. Figures 1g–1ishow a close-
up of this region with a model of an erythromycin mole-
cule (Harris et al., 1965), oriented to show its maximal
dimensions for a size comparison. It can be easily no-
ticed that in the case of the L4 mutant (Figures 1a, 1d,
and 1g), the opening appears smaller (average diameter
?9 A˚at tunnel mouth; ?7 A˚at the point of smallest
constriction; see Figure 4a) than this molecule, while in
the L22 mutant case, the opening is at least twice as
large (?26 A˚maximum). The initiator tRNA-complexed
ribosome displays a conformation in which the mouth
of the tunnel has intermediate size (?20 A˚; Figure 1i).
These values are only approximations because of the
limited resolution, but our measurement of 11 A˚for the
average diameter (using low-pass filtration of the H.
marismortui 50S subunit coordinates to the same reso-
lution as the experimental density maps and a threshold
based on the molecular volume) is in excellent agree-
ment with the actual atomic dimensions (10 A˚) of the
tunnel entrance for H. marismortui (Nissen et al., 2000).
a very similar interior topology (Figure 2d), again shown
as a positive cast in Figure 3 from two different orienta-
tions. Eukaryotic ribosomes were found to exhibit the
same system of tunnels (C.M.T. Spahn, R. Beckmann,
E. Narayanan, J. Helmers, P. Penczek, A. Sali, G. Blobel,
and J.F., unpublished data). The main tunnel, which is
approximately 100 A˚long and can accommodate about
40 amino acids (see Hardesty and Kramer, 2001), pro-
surface ofthe ribosomethat bindsto thebacterial mem-
parts of ribosomal proteins L24 and L29 (Ban et al.,
2000) and was demonstrated to be continuous with the
central pore of the translocon channel in yeast (Beck-
mann et al., 1997; Menetret et al., 2000). One of the
additional routes branching off the main tunnel in the
segment formed by domains I and III of 23S RNA, close
to the main exit, was originally discovered at the 25 A˚
resolution level (Frank et al., 1995). The branching point
is ?70 A˚away from the tunnel entrance. H. marismortui
proteins L15 and L29 are closest to the end of this
second branch (Ban et al., 2000), whose length is 50 A˚.
region, which is the widest (?20 A˚) segment of the main
tunnel, partiallyfollow theextensions ofproteins L4(exit
3) and L22 (exit 4). The lengths of these branches, mea-
sured starting from the common branching point, are
approximately 100 A˚and 40 A˚, respectively.
Effect of the Erythromycin-Resistant Mutations
on the Tunnel Topology
Changes in both proteins carrying erythromycin-resis-
tance mutations (a single amino acid substitution
Lys63Glu in L4 and a deletion of three amino acid resi-
dues, Met82, Lys83, and Arg84 in L22; Chittum and
of their tails extending from the globular parts, specifi-
cally the tip portions (Unge et al., 1998). This is obvious
from the crystal structure of Thermus thermophilus L22
protein, which has high sequence similarity with that of
E.coli.For L4,wederivedthis conclusionindirectlyfrom
for T. maritima (Worbs et al., 2000) and H. marismortui
(Ban et al., 2000) and the sequence homology between
E. coli and T. maritima. The tips of these proteins form
a 12 A˚long and approximately 15 A˚wide segment of
the polypeptide exit tunnel (Ban et al., 2000; Nissen et
The system of tunnels extracted from the densities
obtained from the correspondingly filtered X-ray coordi-
nates of the H. marismortui 50S subunit (yellow in Fig-
ures 3a–3c) is shown together with L4 and L22 proteins
(blue) and overlaid with those 23S RNA residues whose
1999) and with sites protected by erythromycin binding
(green) (Moazed and Noller, 1987). (E. coli numbering
for the affected/protected residues was corrected to
account for the difference in sequence from H. maris-
mortui, whose numbering system was used in Figure 3.)
Tunnel systems for the erythromycin-resistant mutant
ribosomes visualized in this work are shown in Figures
4a and 4b (L4) and Figures 4c and 4d (L22). Besides the
The 50S Subunit Tunnel System
In order to study variations in the tunnel geometry, we
culated on the basis of molecular volume estimates. It
immediately became evident that there exists an entire
system of tunnels. Apart from the main tunnel, three
resulting from holes and cavities in the volume, thereby
creating one continuous branched mass. This proce-
dure left only the passages connected to the main tun-
nel, which therefore represent the more extended fea-
tures of the tunnel system. Figure 2a displays the 50S
stration purposes. Mass corresponding to the inverted
empty space of the volume shown represents a system
of four tunnels (shown in yellow in the cut ribosome
presented in Figure 2b). Filtered uniformly to 17 A˚, all
available cryo-EM maps of E. coli ribosomes display on
average a very similar pattern (Figure 2c). X-ray coordi-
Figure 2. Initiator tRNA-Complexed E. coli
Ribosome 50S Subunit and Tunnel System
(A) 50S subunit cut in the plane along the
main tunnel, shown with the cutting surface
in white, and with the fMet-tRNAfMetmasked
off the map.
(B) Same as (A), with tunnel cast shown in
and displayed in the same view, as “positive
cast,” (C) for the initiation-like ribosomal
complex and (D) for the X-ray structure of
the H. marismortui 50S subunit. IC, interface
canyon, painted in lighter yellow (here and in
Figures 3 and 4) to distinguish it from the
connected tunnel system. Other landmarks
are labeled as in Figure 1. The location of
section where tunnel entrance width was
measured is marked with red arrows. Scale
bar ? 30 A˚.
big difference in the entrance part of the tunnel system
(formed by the central loop of domain V in 23S RNA),
the appearance of an additional cavity in the putative
mutant (circled in red). The cavity, observed as extra
to the tip of this protein (as seen from Figures 2c and
2d) and might be formed as a result of the mutation in
the tip. It possibly involves reorganization in the central
loop system of domain V, induced by a transition in helix
35 of domain II.
Spahn et al., 1999) can be roughly classified into two
groups. The first is comprised of those with small (ap-
proximately 10 A˚) opening of the tunnel (some of the
ribosomes without any ligand bound, including the 30S
subunit helix 27 mutants [Gabashvili et al., 1999] and
the L4 mutant). The unbound 50S subunit structure ob-
tained by X-ray (Ban et al., 2000) and cryo-EM studies
(Matadeen et al., 1999; Mu ¨ller et al., 2000) also falls into
the category with narrow opening (?10 A˚). The second
group, consisting mainly of ribosomes in an active com-
plex, including those with tRNAs and EF-G molecule
bound, have a wider opening (16–20 A˚). It is interesting
that in the last case, the stalk portion of the ribosome
is usually very well defined, which allows us to conclude
that it is in a certain “fixed” conformational state (Agra-
wal et al., 1999). Figure 4 displays tunnel systems for
two ribosome reconstructions representative for these
two groups (Spahn et al.,1999; Agrawal et al., 1999).
Effect of Certain Ligands (tRNA and EF-G)
and Mutations on the Tunnel System
and the Overall Topology
Tunnels in the investigated cryo-EM volumes of the E.
coli 70S ribosome (Agrawal et al., 1998, 1999, 2000,
2001; Gabashvili et al., 1999, 2000; Malhotra et al., 1998;
Figure 3. The H. marismortui Tunnel System
and Its Spatial Relationship with Mutation
and Protection Sites
The tunnel system (yellow) is shown in two
L4 and L22 (blue); RNA nucleotides are per-
turbed as a result of mutation in these proteins
(red; Gregory and Dahlberg, 1999), erythro-
mycin protection sites (green; Moazed and
Noller, 1987), and (C) RNA chains (shown in
Ribbons; Carson, 1997) extended from the
tunnel to the stalk base region (dark blue,
nucleotides 2052–2110, a region connecting
domains IV and V; magenta, nucleotides
2479–2577, central loop system of domain V; purple, nucleotides 1128–1234, RNA component of the stalk base). Asterisk (*) marks A2486
(hidden in [A]) of the peptidyl-transferase center (H. marismortui numbering; Ban et al., 2000; Nissen et al., 2000).
Gating Mechanisms of the Polypeptide Tunnel System
Figure 4. Tunnel Systems of the 50S Subunit of Mutants and Wild-Type
The tunnels are shown in the same views as in Figure 3.
(A and B) L4 mutant.
(C and D) L22 mutant.
(E and F) 70S ribosome used as one of the wild-type controls (Spahn et al., 1999).
(G and H) 70S ribosome complexed with EF-G in its GTP state (Agrawal et al., 1999). Arrows mark the site of the tunnel entrance. The red
dashed circles in (C) and (D) show the putative site of interior erythromycin binding. The location of section where tunnel entrance width was
measured is marked with red arrows. The tunnel systems of the 50S ribosomal subunit depicted here can be viewed in three dimensions at
Another interesting observation can be made regard-
ing the side tunnels, especially the one leading to exit
2. This extension of the main tunnel was observed to
resolution ones (25 A˚; Frank et al., 1995). In contrast, it
is blocked in both erythromycin-resistant mutants (ap-
parent as disrupted densities in Figure 4). This could be
caused by changes in the tertiary structure of domain I
forming this path and contacting both L4 and L22 pro-
teins. The blockage could involve part of its central loop
region (between helices 4 and 5), helices 7 and 18.
that a conformational change in the central protuber-
ance (E. coli G2351, located in its base on the L1 protein
side) might be caused by the L22 mutation as well.
In the present work, all major long-range changes in
morphology were detected in regions of the interface
canyon and central protuberance. Another interesting
distant effect involving stabilization of the extended
stalk might be explained by the transmission of an allo-
steric signal through closely packed regions of the 23S
RNA. It would involve the single-stranded region con-
necting domains IV and V (nucleotides 2052–2110, H.
marismortuinumbering, showninFigure 4cin darkblue)
and contacting both L4 and L22 proteins. Single-
stranded RNA forming the central loop of domain V
builds the neighboring part of the tunnel and its en-
trance. It connects the peptidyl-transferase center and
the A-site tRNA binding regions (colored in magenta in
Figure 3c). The RNA component of the stalk base starts
in the immediate vicinity (purple in Figure 3c; Ban et al.,
The main effect of the mutations is probably due to
the change in electrostatic potential and, thus, in the
ribosomal scaffolding proteins. In the case of L4, a
strongly basic residue is substituted by an acidic amino
acid residue. In the case of L22, two of the three deleted
amino acids well conserved among the species are also
would affect protein-RNA recognition and could easily
Observed Changes of Tunnel Geometry Support
Previous Biochemical and Genetic Data
Proteins L4 and L22 are known to be among the early
assembly proteins of the large subunit (Herold and Nier-
haus, 1987; Stelzl et al., 2000). These proteins play a
scaffolding role, and their modifications could perturb
the assembly of the 50S particle, resulting in large-scale
deformations. Expected changes might involve the to-
pography of the 5S RNA region (central protuberance),
as L4 protein is needed for the correct assembly of this
region (Rohl and Nierhaus, 1982) and interactions of
macrolide antibiotics and the 5S RNA with the peptidyl-
transferase region were shown to have functional impli-
ing study (Gregory and Dahlberg, 1999) demonstrated
perturb the assembly of the 50S ribosomal subunit to-
ward an alternative conformation.
would be important as a means to hold the polypeptide
chain in a defined position during the peptide bond syn-
thesizing reaction. In contrast, opening of the tunnel
wouldbeneeded toallowthemovement ofthepolypep-
tide and to provide space for the next amino acid to be
added. An indication for such dynamic behavior may be
seen from the comparison of two cryo-EM maps of E.
coli ribosomes (Figures 2c and 4e) that are derived from
fMet-tRNA·ribosome complexes or empty ribosomes,
respectively: In the first, the entrance was measured as
20 A˚and in the other as 11 A˚.
L4 and L22 mutant ribosomes, visualized in the pres-
ent study, might be impaired in the dynamic capabilities
of the tunnel, being permanently locked in one of two
extreme conformational states, or they might oscillate
around different average conformations. In this respect,
which found the L4 mutant ribosomes to be functionally
defective (several-fold reduced rate of peptidyltransfer-
ase activity), while the L22 mutant ribosomes exhibited
close to normal activity rates, are of interest.
Communication with the 30S subunit would be very
important in the gating of the tunnel to coordinate the
movement of mRNA with the movement of the nascent
polypeptide chain. Evidence for the existence of such
communication comes from the observation that eryth-
romycin binding resistance caused by the modification
of L4 protein can be masked by mutations affecting S5
mRNA reading abilities to the ribosome (Saltzman et al.,
1974; Saltzman and Apirion, 1976; Alksne et al., 1993).
Thus, conformational changes in the 30S subunit ob-
served in the present work might be synchronized with
the dynamic opening and closing of the tunnel through
a signal passed between the subunits.
Erythromycin and the Tunnel System
Active components of the erythromycin binding sites
are located in domains V and II of 23S rRNA, in the
immediate vicinity of the ribosomal tunnel (Hansen et
al.,1999). The major effects of erythromycin are strong
protections at A-2058, A-2059, and G-2505 of 23S RNA,
as was demonstrated by dimethyl sulfate and kethoxal
probing by Moazed and Noller (1987). These sites (in
the H. marismortui numbering) are shown in green in
Figure 2. Erythromycin binding regions are overlapping
with those from many other antibiotics (for example,
Moazed and Noller, 1987; Nissen et al., 2000), but not
with tRNAs. They are all effective at similar ribosomal
stoichiometries (Mazzei et al., 1993; Sakakibara and
Omura, 1984). Erythromycin resistance mutations in L4
and L22 proteins affect the conformation of nucleotides
in domains II, III, and V of 23S rRNA, which are located
the entrance of the tunnel and the peptidyl-transferase
center. Mapping of these residues (shown in red on
Figure 3)known froma chemicalprobing study(Gregory
and Dahlberg, 1999) can be done based on homology
models for E. coli derived from X-ray coordinates of H.
marismortui ribosomal 50S subunit (Ban et al., 2000)
and E. coli 23S RNA models (Mu ¨ller et al., 2000). RNA
residues mutated in macrolide-resistant ribosomes are
located in the same region (for example, Douthwaite
and Aagaard, 1993; Tait-Kamradt et al., 2000). These
observations are in line with the opinion that erythromy-
(Hardesty and Kramer, 2001), that is, inside the ribo-
The results of the present work favor this interpreta-
tion. The smaller cross-section of the tunnel entrance
would significantly decrease or entirely abolish the
erythromycin-penetrating capabilities for the L4 mutant,
whereas the enlarged cross-section and the formation
of a concavity between the beginning of the tunnel and
the tip of protein L22 would render the binding of eryth-
romycin to the ribosome of the L22 mutant ineffective.
We note that a polypeptide chain is approximately 6 A˚
in diameter, whereas the erythromycin A molecule, al-
though partially flat, slightly exceeds 15 ? 10 A˚. This
explains why blocking or reducing the penetrability for
Generally, the action of an antibiotic might be due to
its presence on the ribosome (physical blocking) or due
to the conformational alterations triggered by its bind-
ing. Our observations of the changes in the mouth of
the tunnel in erythromycin-resistant mutants suggest
that the action of erythromycin falls in the first category.
Topology of the Nascent Chain Transport
inside the Ribosome
From the foregoing, the tunnel system traversing the
large ribosomal subunit appears to be not merely a pas-
sive conduit and a travel convenience for the newly
synthesizing polypeptide chain, but rather an active dy-
namic transport facility that might be capable of stop-
ping and regulating the traffic. Nascent proteins are ei-
ther cotranslationally integrated into a specific complex
located to another destination. The first chain of amino
acids (an N-terminal stretch of 20 or 30 hydrophobic
amino acids) might be a signal sequence initially recog-
nized and sensed from inside the ribosome to trigger
reactions determining the further destination of the pro-
tein (Liao et al., 1997). This regulation would require
dynamic changes, in particular the sequential closing
and opening of the lumenal and cytosolic ends of the
transmembrane pore (Liao et al., 1997; Mu ¨ller et al.,
2001). Besides, certain amino acids of preproteins can
be cotranslationally modified by specific enzymes. Vari-
ous other factors involved in targeting and folding asso-
ciated with the ribosome must gain access to the na-
scent chain during translocation.
The main tunnel is the shortest route and the most
logical way out of the ribosome. Although almost all
cross-links between the nascent polypeptide and ribo-
Mouth of the Tunnel System May Act
as an Intrinsically Dynamic Gate
Acorrelation ofcertainfunctional stateswith thetopolo-
gies of the tunnel system observed in the erythromycin-
resistant mutants points to a possible connection be-
and close states of the tunnel. Closing of the entry site
Gating Mechanisms of the Polypeptide Tunnel System
Both reconstructions were filtered to the same resolution of 17 A˚.
chosen based on molecular volume estimates. For H. marismortui,
the absence of several proteins and RNA fragments in the X-ray
structure was taken into account in the molecular volume estimate.
All image processing steps were performed using the SPIDER
image processing system (Frank et al., 1996) on the Origin 2000
(Silicon Graphics, Mountain View, CA).
somal RNA track the common path of the tunnel system
91 in E. coli) was located closer to the main exit (Choi
and Brimacombe, 1998). Why does the ribosome need
additional routes branching off the main pathway near
the main exit? One possibility is that these openings
could maintain the necessary chemical equilibrium in
the tunnel system, providing access for water and ion
molecules. Another is that they could be used for a
more complex regulation of peptide translocation and
a nascent peptide passes the gate created by exten-
sions of proteins L4 and L22. This gate might work as
a sensor, causing conformational rearrangements de-
pending on the nature of the polypeptide passing
through it (as suggested by Nissen et al., 2000). We
speculate that L4 and L22 proteins, apart from partici-
pating in thegating of the entrance ofthe tunnel system,
may also regulate the use of alternative exits. This regu-
lation could cooperate with cotranslational protein pro-
cessing or might even be responsible for the choice of
the final destination for the nascent polypeptide chain.
After completion of this work, the X-ray structure of
the 70S ribosome from T. thermophilus based on a 5.5 A˚
electron density map was published (Yusupov et al.,
tRNAs bound in the P and E sites. We used the publicly
deposited coordinates of rRNA to obtain a map with the
same resolution as used in our study and found a tunnel
system with very similar topology as in E. coli and H.
marismortui described here, although the tunnel en-
trance appearsmore narrow(?5 A˚).Since theresolution
of the map was insufficient to allow remodeling of the
proteins, it is not possible to draw conclusions about
the role of the tip portions of these proteins in defin-
ing the ribosome tunnel in T. thermophilus. However,
given the high similarity of the tunnel topology in all
three species, it is likely that the same mechanisms of
gating will apply.
A homology model of L4 protein was built based on the structural
alignment of C-? atom positions deposited in Protein Data Bank for
H. marismortui (Ban et al., 2000), all-atom coordinates for the globu-
lar part of this protein from T. maritima (Worbs et al., 2000), and
overall sequence alignments (BLAST) for T. maritima and E. coli.
Modeling was performed on a Silicon Graphics using Homology
and Discover modules of the software Insight II (version 99) from
This work was supported by grants NIH R37 GM29169, R01
GM55440, and P41 RR01 219 and NSF BIR 9219043 (to J.F.) and
NIH R01 GM19756 (to A.E.D.). We are grateful to Ki H. Kim from
romycin A molecule, Christian Spahn for helpful comments and
discussion, and Yu Chen and Michael Watters for assistance with
Received March 13, 2001; revised May 1, 2001.
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