Correction for Berk et al., Structural basis for mRNA and tRNA positioning on the ribosome
- SourceAvailable from: Anton Olegovich Chugunov[show abstract] [hide abstract]
ABSTRACT: Ribosomal protein S2 is an essential component of translation machinery, and its viable mutated variants conferring distinct phenotypes serve as a valuable tool in studying the role of S2 in translation regulation. One of a few available rpsB mutants, rpsB1, shows thermo-sensitivity and ensures enhanced expression of leaderless mRNAs. In this study, we identified the nature of the rpsB1 mutation. Sequencing of the rpsB1 allele revealed a G to A transition in the part of the rpsB gene which encodes a coiled-coil domain of S2. The resulting E132K substitution resides in a highly conserved site TKKE, a so-called N-terminal capping box, at the beginning of the second alpha helix. The protruding coiled-coil domain of S2 is known to provide binding with 16S rRNA in the head of the 30S subunit and, in addition, to interact with a key mRNA-binding protein S1. Molecular dynamics simulations revealed a detrimental impact of the E132K mutation on the coiled-coil structure and thereby on the interactions between S2 and 16S rRNA, providing a clue for the thermo-sensitivity of the rpsB1 mutant. Using a strain producing a leaderless lacZ transcript from the chromosomal lac-promoter, we demonstrated that not only the rpsB1 mutation generating S2/S1-deficient ribosomes, but also the rpsA::IS10 mutation leading to partial deficiency in S1 alone increased translation efficiency of the leaderless mRNA by about 10-fold. Moderate overexpression of S1 relieved all these effects and, moreover, suppressed the thermo-sensitive phenotype of rpsB1, indicating the role of S1 as an extragenic suppressor of the E132K mutation.Journal of bacteriology 10/2012; · 3.94 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Here we present analysis of a 3D cryo-EM map of the 70S ribosome from Mycobacterium smegmatis, a saprophytic cousin of the etiological agent of tuberculosis in humans, Mycobacterium tuberculosis. In comparison with the 3D structures of other prokaryotic ribosomes, the density map of the M. smegmatis 70S ribosome reveals unique structural features and their relative orientations in the ribosome. Dramatic changes in the periphery due to additional rRNA segments and extra domains of some of the peripheral ribosomal proteins like S3, S5, S16, L17, L25, are evident. One of the most notable features appears in the large subunit near L1 stalk as a long helical structure next to helix 54 of the 23S rRNA. The sharp upper end of this structure is located in the vicinity of the mRNA exit channel. Although the M. smegmatis 70S ribosome possesses conserved core structure of bacterial ribosome, the new structural features, unveiled in this study, demonstrates diversity in the 3D architecture of bacterial ribosomes. We postulate that the prominent helical structure related to the 23S rRNA actively participates in the mechanisms of translation in mycobacteria.PLoS ONE 01/2012; 7(2):e31742. · 3.73 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Cryo-EM analysis of a wild-type Escherichia coli pretranslocational sample has revealed the presence of previously unseen intermediate substates of the bacterial ribosome during the first phase of translocation, characterized by intermediate intersubunit rotations, L1 stalk positions, and tRNA configurations. Furthermore, we describe the domain rearrangements in quantitative terms, which has allowed us to characterize the processivity and coordination of the conformational reorganization of the ribosome, along with the associated changes in tRNA ribosome-binding configuration. The results are consistent with the view of the ribosome as a molecular machine employing Brownian motion to reach a functionally productive state via a series of substates with incremental changes in conformation.Proceedings of the National Academy of Sciences 03/2012; 109(16):6094-9. · 9.74 Impact Factor
Structural basis for mRNA and tRNA positioning
on the ribosome
Veysel Berk*, Wen Zhang†, Raj D. Pai*, and Jamie H. D. Cate*†‡§
Departments of *Molecular and Cell Biology, and†Chemistry, University of California, Berkeley, CA 94720; and‡Physical Biosciences Division,
Lawrence Berkeley National Laboratory, Berkeley, CA 94720
Communicated by Harry F. Noller, University of California, Santa Cruz, CA, August 29, 2006 (received for review July 24, 2006)
Protein synthesis requires the accurate positioning of mRNA and
tRNA in the peptidyl-tRNA site of the ribosome. Here we describe
x-ray crystal structures of the intact bacterial ribosome from
Escherichia coli in a complex with mRNA and the anticodon stem-
loop of P-site tRNA. At 3.5-Å resolution, these structures reveal
rearrangements in the intact ribosome that clamp P-site tRNA and
mRNA on the small ribosomal subunit. Binding of the anticodon
stem-loop of P-site tRNA to the ribosome is sufficient to lock the
head of the small ribosomal subunit in a single conformation,
thereby preventing movement of mRNA and tRNA before mRNA
x-ray crystallography ? protein synthesis
each peptide bond is made, the ribosome must rearrange its
contacts with mRNA and tRNA to allow translocation along the
mRNA by a single 3-nt codon, with a concomitant movement
of the cognate tRNAs. The ribosome controls the positioning of
mRNA and tRNAs during translation through a number of
direct intermolecular contacts (1). However, the molecular
mechanisms by which the ribosome balances maintenance of the
mRNA reading frame with rapid translation remain unclear (2).
Of the three tRNA binding sites on the ribosome [aminoacyl
(A), peptidyl (P), and exit (E)], tRNA binds most tightly to the
peptidyl-tRNA site (P site) (3), where the reading frame on the
mRNA is maintained (4). Notably, the anticodon stem-loop
(ASL) of tRNA, which binds in a cleft in the small (30S)
ribosomal subunit, contributes a significant amount to the free
energy of binding to the P site (5) in an mRNA-dependent
manner (6, 7). The interactions among mRNA, tRNA, and the
ribosome in the P site are only approximately known, having
been determined at ?5-Å resolution from x-ray crystal struc-
tures of the 70S ribosome (8). Structures of the 30S subunit at
atomic resolution contain mimics of mRNA and the ASL of
P-site tRNA that rely on crystal contacts between neighboring
molecules (1, 9). However, the mRNA mimic does not form
Watson–Crick base pairs to the ASL analog, and the ASL lacks
a canonical 7-nt anticodon loop structure. We have now deter-
mined two structures of the intact 70S ribosome from Esche-
richia coli in a complex with mRNA and the ASL of initiator
tRNAfMet(Fig. 1A) at a resolution of 3.5 Å (Fig. 1B and Table
1). These structures provide a more detailed view of how the
ASL of P-site tRNA interacts with the intact ribosome.
rotein synthesis, or translation, requires the ribosome to hold
tRNAs in one reading frame of the mRNA. However, after
Results and Discussion
Overall Conformation of the Ribosome.Inthepresent70Sribosome
structures, both ribosomes in the crystallographic asymmetric
unit bind mRNA and the ASL to the P site of the 30S subunit
in an identical manner. We therefore averaged the electron
density maps of both ribosomes to improve the effective reso-
lution of the maps to ?3.5 Å (see Materials and Methods). Given
the nearly identical conformations of large portions of the two
ribosomes, we describe the interactions in one of the ribosomes
as representative of both. The crystals used to determine the
present structures were grown in conditions highly similar to
those reported previously (10). Therefore, we were able to
compare the present structures to the apo-70S ribosome struc-
tures to determine the effects of mRNA and ASL binding to the
ribosomal P site. Surprisingly, binding of mRNA and the P-site
ASL completely reverses the swiveling of head of the 30S subunit
observed in the previous structures in the absence of ligands
(Fig. 1C) (10). In contrast, there is no appreciable change in the
global conformation of the large subunit that can be attributed
to ASL binding. When compared with lower-resolution struc-
tures of the 70S ribosome, the conformation of the ribosome in
the present structures adopts a pretranslocation conformation
(2, 8). These structural results indicate that in the intact ribo-
some the position of the head of the small subunit is inherently
dynamic in the absence of bound ligands and is free to rotate
about the neck helix in 16S rRNA (10). The head of the small
subunit is only stabilized in a single conformation upon binding
mRNA and the ASL portion of P-site tRNA. In agreement with
this idea, the neck helix in human ribosomes has also been
observed to be dynamic in the absence of bound ligands (11).
Position of mRNA in the 30S P Site.Inthe70Sstructures,themRNA
is held in place entirely by interactions with its phosphoribose
backbone. This mode of binding contrasts with the ribosomal
stabilize the codon–anticodon helix (9). The mRNA is threaded
in nearly the same conformation as was observed in the 30S
subunit structures (9). The backbones of all three nucleotides in
the P-site codon are coordinated by hydrogen bonds to univer-
sally conserved nucleotides in 16S rRNA (12). The phosphate of
nucleotide ?1 is within hydrogen-bonding distance of the exo-
cyclic amine (N2) of G926 in 16S rRNA (Fig. 2A), in agreement
with protection of this nucleotide from chemical probes when
P-site tRNA is bound (13). Nucleotide m3U1498 in 16S rRNA
makes a number of contacts to the backbone of nucleotides ?1
and ?2. The phosphate of mRNA nucleotide ?2 is within
hydrogen-bonding distance of the ribose 2?-OH of m3U1498 in
16S rRNA, whereas the 2?-OH of nucleotide ?1 hydrogen-bonds
nucleotide ?2 is stacked on the base of m3U1498 (Fig. 2A). Two
cytidines, m4Cm1402 and C1403, hydrogen-bond via their N4
amines to the nonbridging phosphate oxygens of mRNA nucle-
otide ?3 (Fig. 2A).
In addition to the above rRNA contacts, a single metal ion is
coordinated to the major groove of the mRNA–ASL codon–
Author contributions: V.B. and J.H.D.C. designed research; V.B., W.Z., R.D.P., and J.H.D.C.
performed research; V.B., W.Z., R.D.P., and J.H.D.C. analyzed data; and V.B., W.Z., R.D.P.,
and J.H.D.C. wrote the paper.
The authors declare no conflict of interest.
Abbreviations: P site, peptidyl-tRNA site; ASL, anticodon stem-loop.
Data deposition: The atomic coordinates and structure factors have been deposited in the
Protein Data Bank, www.pdb.org (PDB ID codes 2I2P, 2I2T, 2I2U, and 2I2V).
§To whom correspondence should be addressed. E-mail: email@example.com.
© 2006 by The National Academy of Sciences of the USA
October 24, 2006 ?
vol. 103 ?
anticodon helix (9). This specifically bound ion was not present
in the apo-70S ribosome structures (10). The position of the
density is most consistent with a fully hydrated Mg2?ion, with
the Mg2?positioned ?3.5 Å from the N7 and O6 of universally
conserved nucleotide G1401 (12), which has been shown to be
critical for mRNA binding (9, 14). Notably, all six of the modeled
water ions coordinating the Mg2?are within hydrogen-bonding
distance of the major groove face and phosphate of G1401 in 16S
rRNA and the phosphate oxygens of nucleotides ?2 and ?3 in
the mRNA (Fig. 2B).
but does not form a base pair (3-methyl-uridine, m3U, in E. coli),
packs against the phosphate and ribose of mRNA nucleotide ?2,
as described above (Fig. 2A). The base of m3U1498 also stacks
on another universally conserved nucleotide, G1497, and creates
an electronegative pocket at the top of helix 44 immediately
adjacent to the phosphate of mRNA nucleotide ?3 and the
phosphates of 16S nucleotides 1494–1496. Intriguingly, this
pocket is occupied by electron density that runs parallel to the
entire P-site codon, with one end stacking on base A790 in 16S
rRNA and the other approaching the position where mRNA
nucleotide ?4 would be positioned (Fig. 2C). Structural refine-
ment revealed that the electron density corresponds to nucleo-
tides ?4 through ?6 in the short mRNA, along with two Mg2?
ions (Fig. 2D). One of the Mg2?ions is coordinated to the
phosphate of nucleotide ?6 and is juxtaposed to the major
groove face of G1497 and m3U1498, whereas the other bridges
the phosphates of mRNA nucleotide ?5 and 16S rRNA nucle-
The tight folding of the 3? end of the short mRNA into the
pocket formed by A790, the top of helix 44, and the codon–
anticodon interaction in the P site comes as a surprise. As noted
above, all of the residues lining this pocket are universally
conserved (12). The combination of an exposed purine base
(A790) and the electronegative pocket within which a Mg2?ion
is coordinated (G1497 and m3U1498 and the phosphates of
nucleotides 1496–1498) suggests that the site occupied by
mRNA nucleotide ?6 could bind free nucleotides in certain
circumstances. A number of cyclic nucleotides that serve as
second messengers (cAMP, cGMP, and cyclic diguanylate) are
used in bacteria and eukaryotes to regulate cell physiology (15,
codon–anticodon base pairing. Numbering of mRNA and ASL nucleotides is indicated. (B) Anisotropic diffraction from crystals of the 70S ribosome complexed
with a P-site ASL and mRNA. Completeness of the data as a function of resolution is plotted on the right axis, and the mean signal-to-noise of diffraction
atoms (light blue) and C? atoms (dark blue). In the ribosome complex, the ASL and mRNA are shown as space-filling models, and the 5? to 3? direction of mRNA
of the ribosome are labeled for the 30S head (Head), and 50S central protuberance (CP), as are ribosomal proteins in the small (S) and large (L) subunits.
Overall structure of intact E. coli 70S ribosome in a complex with mRNA and P-site ASL. (A) Secondary structures of the ASL and mRNA–ASL
Table 1. X-ray crystallographic statistics and refinement
a, b, c, Å
No. of unique reflections
Torsional dynamics refinement
No. of reflections
No. of atoms
211.8, 395.2, 744.4
200 to 3.22
Berk et al. PNAS ?
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vol. 103 ?
no. 43 ?
16). It will be interesting to test whether these nucleotides can
regulate translation by binding to this site.
Positioning of the P-Site ASL. In contrast to the pyrimidine?
pyrimidine base pairs seen in the 30S crystal structures (9), the
wider codon–anticodon helix formed between the mRNA and
P-site ASL pulls the ASL ?2 Å deeper into the P-site binding
pocket. In addition, the binding pocket is ?1.3 Å more closed
deeper binding of the ASL in the P site leads to stacking of the
mRNA in the P site. (A) Hydrogen bonds to the phos-
and a fully hydrated Mg2?have been removed for
clarity. (B) Coordination of a fully hydrated Mg2?to
perspective of the subunit body. (C) View of (Fobs?
pocket between the backbone of P-site mRNA and
helix-44 nucleotides 1494–1498 in 16S rRNA. The po-
sition of A1493 is already adjusted to fit the electron
density. (D) Nucleotides ?4 through ?6 of the mRNA,
along with two Mg2?ions, modeled into the electron
density in C followed by refinement.
Interactions between the ribosome and
between C1400 and G966 and between G966 and ASL are indicated by arrows and distances. The position of the very C terminus of protein S9 is indicated. The
view is from the aminoacyl-tRNA site in the small subunit. (B) Stereoview of the averaged (3Fobs? 2Fcalc) difference electron density for the 30S contacts to the
ASL shown in A. Electron density for mRNA nucleotides ?4 through ?6 has been removed for clarity. The density indicated by an asterisk is disconnected from
that for protein S9 and therefore has not been assigned. (C) Minor groove interactions among G1338, A1339, and the P-site ASL. The view is from the right in
A, i.e., from the perspective of the 50S subunit.
Ribosome interactions with the P-site ASL. (A) Closing of the P-site cleft when compared with the 30S ribosome structures (9). Changes in the distance
www.pnas.org?cgi?doi?10.1073?pnas.0607541103 Berk et al.
ribose and base of nucleotide 34 in the anticodon with both G966
and C1400 in 16S rRNA, respectively. Both of these nucleotides
in 16S rRNA have been directly implicated in P-site tRNA
binding (14, 17). Notably, deeper binding of the ASL in the P site
does not appreciably change the interactions between the 30S
subunit and the ASL that clamp the P-site tRNA in position
closer to the small and large (50S) subunit interface. These
contacts involve universally conserved nucleotides A790 on the
platform and nucleotides G1338 and A1339 in the 30S head (9).
in the platform of the small subunit, forms a hydrogen bond to
the 2?-OH of the ribose of ASL nucleotide 38. As observed in the
30S subunit crystal structures, G1338 forms a type II ‘‘A-minor’’
interaction with nucleotide 41 of the ASL (9, 18). A1339 forms
a type I A-minor motif with the G30–C40 base pair of the ASL
(Fig. 3C) (18).
Two ribosomal proteins in the small subunit, S13 and S9, have
been shown in Thermus thermophilus 30S subunit structures to
interact with P-site tRNA through their C termini (9). Although
neither the C terminus of S13 nor the C terminus of S9 is
essential for viability (19, 20), their presence may contribute to
the efficiency and selectivity of translation initiation (21). In the
E. coli ribosome neither protein has a highly ordered C terminus
when compared with other proteins in the 30S subunit. The C
terminus of S13, which is 10 aa shorter than in T. thermophilus,
extends toward the minor groove of the top three base pairs of
the ASL (Fig. 1A). However, the C-terminal 5 aa of protein S13,
including three lysines, are disordered in the present structures.
In E. coli, protein S13 therefore does not make specific contacts
to the ASL part of P-site tRNA but may simply contribute
electrostatic stabilization to P-site tRNA binding. The highly
conserved C terminus of protein S9, for which only the backbone
is visible in the present structures, points toward the major
groove face of the RNA U-turn at the tip of the anticodon loop
of the C-terminal residues of protein S9 are not well ordered
suggests that protein S9 may also contribute primarily electro-
static interactions with P-site tRNA.
Closing of the 30S Head Domain. Although the above contacts
among the ASL, mRNA, and 30S subunit are similar to those
reported previously in the 30S structures (9), the position of the
small subunit head leads to a different positioning of the ASL
with respect to the intact ribosome. In the present structures the
70S ribosome adopts a closed conformation of the 30S head
when compared with isolated 30S subunit structure with an
empty aminoacyl-tRNA site (9). This closing involves a tilting of
the 30S head toward the central protuberance of the 50S subunit
(Fig. 4). Closing of the small subunit head seen in the present
structures is consistent with rearrangements in the small subunit
observed in solution upon subunit association (22).
Based on the ASL analog position in the 30S subunit struc-
tures, an intact tRNA projected from its orientation would clash
with the central protuberance of the large ribosomal subunit. In
contrast, the ASL observed in the 70S ribosome structures would
extend the P-site tRNA in the correct direction to dock in the
P-site cleft without these clashes (Fig. 4). This difference in ASL
positioning may explain the need for closing of the head domain
when the 30S subunit docks with the 50S subunit. Tilting of the
30S head domain toward the central protuberance in the 50S
subunit also results in closing of the P-site cleft (Fig. 3 A and B).
Closing of the P-site cleft in the absence of A-site tRNA likely
increases the number of stabilizing interactions between the
codon–anticodon helix in the P site and the ribosome, i.e., with
G966. This closing may therefore help to minimize frame-
shifting on the mRNA before mRNA decoding when the ami-
noacyl-tRNA site is empty.
Materials and Methods
Ribosome Crystallization and Diffraction Data Measurement. Ribo-
somes were purified from E. coli strain MRE600 as reported
(10), with slight modifications. S1 was depleted from intact 70S
ribosomes by using a PolyU Sepharose column (23). Eluted
ribosomes were concentrated by pelleting in a Beckman Ti45
rotor (Beckman Coulter, Fullerton, CA) at 158,000 ? g for 8 h.
mRNA (5?-pAUGUUU-3?) from Dharmacon (Lafayette, CO)
were used in 5- to 10-fold excess over ribosomes in crystallization
drops. Ribosomes were crystallized at 4°C by using microbatch
96-well plates and buffers containing 11% 2-methyl-2,4-
with the 30S subunit structure. The structure of Protein Data Bank entry 1J5E
was used for comparisons (9). The direction of closing of the small subunit
head is indicated by an arrow; i.e., the axis of rotation is perpendicular to the
The position of full-length tRNA in the P site as modeled based on the ASL
positions is shown in outline form, with the arrow indicating the change in
direction in going from the 30S to 70S structures.
Closing of the 30S head domain in the 70S ribosome when compared
Table 2. Atoms used in ribosome and tRNA superpositions
Ribosomal domainModels* Atoms used
Intact 30S subunit 70S?mRNA?ASL v. 1J5E?ASL and
70S?mRNA?ASL v. apo-70S, I or II
P atoms of 16S rRNA residues: 9, 14, 20, 21, 23, 125, 296, 297, 299, 558,
561–565, 570, 571, 574, 757, 766, 780, 782, 804, 815–818, 820–822, 825, 861,
864, 865, 867, 869, 873, 875–879, 881, 883, 905, 916, 917, 920, 921, 1068,
1077, 1078, 1081, 1393, 1394, 1501, 1509, 1510, 1524, 1525 (see ref. 30)
C1? of 28–42P-site tRNA 70S?mRNA?ASL v. 1GIX, chain I
and 1J5E?ASL v. 1GIX, chain I
Berk et al. PNAS ?
October 24, 2006 ?
vol. 103 ?
no. 43 ?
pentanediol, 2% PEG 8000, 27 mM MgCl2, 255 mM NH4Cl, 125
mM KCl, 1 mM spermine, 0.5 mM spermidine, 10 mM Tris (pH
7.5), 0.25 mM EDTA, and 3.5 mM 2-mercapthoethanol. These
conditions are similar to those used previously with apo-70S
ribosomes (10). Ribosome crystals were stabilized with crystal-
lization buffer containing additional 2-methyl-2,4-pentanediol,
PEG 400, and MES (pH 7.0) to allow cryocooling of the crystals
to liquid nitrogen temperatures. Diffraction data were measured
from 13 crystals cooled to 100 K by using 0.25° to 0.3° oscillations
at the SIBYLS (12.3.1) beamline at the Advanced Light Source.
The crystals diffracted anisotropically to 3.2 Å (Fig. 1B). Data
were reduced by using Denzo?Scalepack (24) and Truncate (25),
yielding the statistics shown in Table 1.
Molecular Replacement and Structure Refinement.Thetwocopiesof
the 70S ribosome in the crystallographic asymmetric unit were
positioned by using rigid-body refinement of the apo-70S ribo-
some structures, followed by rigid-body refinement of domains
within the ribosome: 30S head, 30S body, 30S platform, 50S
body, the L1 arm, and the L11 arm (10). Density-modified
electron density maps were generated by using the program
Pirate (26). CNS was used to generate (Fobs? Fcalc) and (3Fobs?
2Fcalc) difference electron density maps (27), with the phases
derived from Pirate-based density modification (26). Additional
restraints used in torsional dynamics and used in modeling
magnesium ions are as described in ref. 10. In the structure
refinement it was determined that large portions of the small and
large ribosomal subunits could be restrained by noncrystallo-
most of the two ribosomes, including the entire 30S subunits, it
was possible to average the electron density for the two ribo-
somes (28). Averaging dramatically improved the overall quality
of the electron density maps by removing anisotropic streaking
apparent in the nonaveraged maps. The averaged (3Fobs ?
2Fcalc) difference electron density maps have features consistent
with a resolution of 3.5 Å, as shown in Fig. 3B.
Least-Squares Superpositions. Comparisons to atomic resolution
structures of the isolated 30S subunit, to tRNAs, and to struc-
tural models of the intact ribosome were carried out by least-
squares superposition in the program O (29), generally using
ribose C1? positions in nucleotides. A subset of phosphorous
atoms identified as nearly invariant in position were also used for
comparisons to the T. thermophilus 30S subunit (30). The
full-length P-site tRNA used for superposition with the ASLs
was taken from the T. thermophilus 70S ribosome (8). A full list
of least-squares superpositions of ribosomes and tRNAs is given
in Table 2.
Figure Preparation. Figures were made by using the programs
Ribbons (31) and PyMol (32).
We thank K. Frankel, S. Classen, G. Meigs, and J. Holton for significant
help with data measurement at the SIBYLS beamline at the Advanced
Light Source. We also thank J. Doudna and H. Noller for helpful
comments on the manuscript. This work was funded by National
Institutes of Health Grant GM65050 (to J.H.D.C.), National Cancer
Institute Grant CA92584 (for the SIBYLS beamline), and Department
of Energy Grant DE-AC03-76SF00098 (to J.H.D.C., and for the SIBYLS
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