Structural insights into +1 frameshifting promoted by
expanded or modification-deficient anticodon
stem loops
Tatsuya Maehigashi1, Jack A. Dunkle1, Stacey J. Miles, and Christine M. Dunham2
Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322
Maintenance of the correct reading frame on the ribosome
is essential for accurate protein synthesis. Here, we report
structures of the 70S ribosome bound to frameshift suppressor
tRNASufA6 and N1-methylguanosine at position 37 (m1G37) modification-deficient anticodon stem loopPro, both of which cause the
ribosome to decode 4 rather than 3 nucleotides, resulting in a +1
reading frame. Our results reveal that decoding at +1 suppressible
codons causes suppressor tRNASufA6 to undergo a rearrangement
of its 5′ stem that destabilizes U32, thereby disrupting the conserved U32–A38 base pair. Unexpectedly, the removal of the
m1G37 modification of tRNAPro also disrupts U32–A38 pairing in
a structurally analogous manner. The lack of U32–A38 pairing
provides a structural correlation between the transition from canonical translation and a +1 reading of the mRNA. Our structures
clarify the molecular mechanism behind suppressor tRNA-induced
+1 frameshifting and advance our understanding of the role
played by the ribosome in maintaining the correct translational
reading frame.
near cognate
| processivity
T
he three polymerase reactions of DNA replication, RNA
transcription, and protein translation are essential to life and
all involve a delicate balance between speed and fidelity. The
bacterial ribosome decodes three mRNA nucleotides into a single amino acid at a rate of ∼20 residues/s with high fidelity (103–
104) (1, 2). High translational fidelity requires the strict maintenance of the triplet mRNA reading frame with shifts in either
the 5′ or 3′ direction (positive or negative shifting, respectively)
occurring infrequently (∼1 in 30,000) (3). Upon shifting of the
mRNA, the proteins expressed are nonsense polypeptides and
typically degraded (4). However, frameshifting rates may be
underestimated because of rapid halting of protein synthesis by the
postpeptidyl transfer quality control mechanism, which recognizes
near- and noncognate interactions in the P site (5). In this case, the
identification of such incorrect proteins would be difficult to detect.
Although it was hypothesized almost 50 years ago that the
triplet genetic code was immutable, the identification of mutagen-induced genomic insertions that still yielded the expression
of the correct protein implied otherwise (6–8). This alternative
reading of mRNA indicated that the ribosome had the ability to
decode a nontriplet reading frame under certain circumstances.
It was reasoned that suppressors to these genomic insertions should
be located adjacent to the original mutation site. However, most
identified frameshift suppressors were extragenic; genetic mapping
showed that the majority of these suppressors were nucleotide
insertions, deletions, and modifications in tRNA genes, typically at
the anticodon stem loop (ASL) (9, 10). Over the next several decades, genetic characterization of these suppressors played a large
role in the fundamental understanding of tRNA structure, decoding, and other general features of ribosome function (reviewed
in ref. 11).
The first bacterial +1 suppressor sequenced was tRNASufD
(suppressor of frameshift D), a derivative of tRNAGlyCCC that
www.pnas.org/cgi/doi/10.1073/pnas.1409436111
contained a cytosine insertion in its ASL and decoded an expanded
glycine codon 5′-GGG-G-3′ (inserted guanosine follows the
GGG glycine codon and is preceded by a hyphen; all codons are
denoted 5′ to 3′) (12). Given the Watson–Crick complementarity
between the inserted nucleotide at the anticodon (C) and codon
(G), a 4-bp interaction between the codon and anticodon was
proposed as the mechanistic basis for the +1 mRNA reading
frame (13). Additional evidence to support this hypothesis included the isolation of suppressor tRNAs from both bacteria (7,
12, 14–17) and yeast (18, 19), all containing additional nucleotides in their anticodon loops that had the ability to Watson–
Crick pair with the corresponding expanded mRNA codon. This
4-bp decoding or quadruplet model was further expanded upon
to state that the size of the tRNA anticodon loop dictated the
mRNA length decoded by the ribosome rather than the number
of potential interactions that form between the tRNA–mRNA
pair (20). Subsequently, this elegant model, termed the “yardstick” model, became dogma in textbooks.
However, emerging evidence began to call both models into
question. The first indication that a 4-nt codon–anticodon interaction is not essential for +1 decoding was the identification
of suppressor tRNASufJ (a tRNAThrGGU derivative), which lacks
Watson–Crick complementarity between the inserted nucleotide
in the anticodon stem and the additional residue in the mRNA
(21, 22). The second indication was that mutational studies of
tRNASufD and other +1 tRNA suppressors showed that Watson–
Significance
Biological fitness is dependent on the accurate flow of genetic
information from DNA to mRNA to protein. Breakdown in ribosome translational fidelity is detrimental because of its
central role in the production of proteins. Altering the 3-base
genetic code usually results in the expression of aberrant or
nonsense proteins that are degraded. Here, we describe molecular snapshots of the ribosome in the process of decoding
a 4-base codon by a frameshift suppressor tRNA that results in
a +1-nt shift of the mRNA reading frame. Conformational dynamics of the anticodon stem loop seem to drive remodeling of
the tRNA–mRNA interaction to promote the +1 movement,
which we predict occurs after accommodation of the tRNA
onto the ribosome.
Author contributions: T.M., J.A.D., and C.M.D. designed research; T.M., J.A.D., and S.J.M.
performed research; T.M., J.A.D., and C.M.D. analyzed data; and T.M., J.A.D., and C.M.D.
wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The crystallography, atomic coordinates, and structure factors have been
deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 4L47, 1VVJ, 4L71, 4LEL,
4LFZ, 4LNT, 4LSK, 4LT8, and 4P70).
1
T.M. and J.A.D. contributed equally to this work.
2
To whom correspondence should be addressed. Email: [email protected].
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1409436111/-/DCSupplemental.
PNAS Early Edition | 1 of 6
BIOCHEMISTRY
Edited by Rachel Green, Johns Hopkins University, Baltimore, MD, and approved July 10, 2014 (received for review May 21, 2014)
Crick complementarity between the anticodon and codon was
not essential for suppression of the +1 site (19, 23–26). Moreover, the isolation of +1 suppressors with normal 7-nt anticodon
loops illustrated that factors other than an increased loop size
may influence frameshifting (27, 28).
High-resolution X-ray crystal structures of the 70S ribosome
have provided molecular insights at multiple steps during protein
synthesis (reviewed in ref. 29). Despite these advances, very
few structural studies have been able to provide a molecular understanding of mRNA frameshifting. Here, we investigated the
structural basis for +1 decoding by a well-studied suppressor,
tRNASufA6 (17, 30). Frameshift suppressor tRNASufA6 is a derivative
of tRNAProCGG and contains an inserted guanosine (G37.5) between positions 37 and 38 in the anticodon loop (Fig. 1A).
tRNASufA6 suppresses a mutagen-induced cytosine insertion in the
Salmonella typhimurium histidine operon by decoding CCC-N codon as proline (where N represents any nucleotide) (17, 30) (Fig.
1B). This 4-nt decoding or +1 frameshifting event restores the correct reading frame and reverts a nonsense truncated protein back to
the WT protein. The presence of an N1-methylation at position 37
(m1G37) in tRNASufA6 precludes base pairing with the cytosinerich proline codon and thus, prevents a 4-base codon–anticodon
interaction (30). We investigated both how tRNASufA6 mediates
frameshifting and what role the modification plays in mRNA
frame maintenance by determining nine X-ray crystal structures of
either the ASLSufA6 or ASLPro bound to the Thermus thermophilus
70S A site at resolutions of 2.9–3.9 Å (Tables S1 and S2). Our
results distinguish between the various models of +1 frameshifting
and provide molecular insights into the frameshifting process.
Results
Structures of ASLSufA6 Bound to Three +1 Suppressible Codons in the
Ribosomal A Site. We solved three structures of 70S ribosomes
programmed with ASLSufA6 and mRNAs containing A-site codons
Fig. 1. Frameshift suppressor tRNASufA6 decodes a 4-nt codon to produce a +1
frameshift. (A) Secondary structure representations of the ASLs of tRNAPro
and tRNASufA6 show base pairing to their respective mRNA codons (blue).
The insertion in the anticodon loop of +1 frameshift suppressor tRNASufA6 is
between nucleotides 37 and 38; G37.5 is red, whereas the m1G37 modification is shown in both. (B) Frameshift suppressor tRNASufA6 decodes the
proline codon as CCC-U (blue) rather than the cognate 3-nt proline codon
CCC (0 frame), suppressing the insertion N nucleotide (any nucleotide but
depicted as a U for simplicity) and bypassing a premature stop codon UGA in
the zero frame. The +1 reading of hisD3018 by tRNASufA6 restores the correct
amino acid sequence with Glu (green) read as the next codon.
2 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1409436111
that support +1 frameshifting: CCC-A, CCC-G, and CCC-U
(where the fourth nucleotide is decoded as part of the CCC proline codon) (17) (Figs. 1A and 2A and Table S1). The antibiotic
paromomycin was used in all 70S structures determined to promote better diffraction. Two structures of 70S programmed with
ASLSufA6 decoding the +1 suppressible codon CCC-U were
solved with and without paromomycin, revealing identical structures (Table S1). In all three 70S-ASLSufA6 structures, a canonical
2-nt codon–anticodon interaction forms with no interactions between ASLSufA6 and the fourth nucleotide of the codon (codon
position 7 with numbering beginning with the P-site codon) (Fig.
2A and Fig. S1). Despite the potential for a Watson–Crick base
pair between the ASL wobble nucleotide C34 and the fourth nucleotide in the expanded CCC-G codon, only a 3-nt codon–anticodon interaction is observed (Fig. 2A). Similar to structures of
tRNA decoding cognate mRNAs on the 30S and 70S ribosomes,
Watson–Crick base pair interactions form at the first and second
positions of the codon–anticodon helix (31, 32). Unexpectedly, at
the wobble position, a C•C mismatch base pair between ASL C34
and mRNA position C6 forms containing a single hydrogen bond
between the N4 amino group of C34 and the N3 atom of C6 of the
mRNA (Fig. 2 A and B). The C34•C6 mismatch pairing is a similar
width as a Watson–Crick C-G base pair (measured from Cl′ to
C1′) (Fig. 2B) and preserves the base-stacking interactions with
adjacent codon–anticodon nucleotides (Fig. 2A). Additionally,
the C34•C6 mismatch contains a similar geometry as an ASLmodified cmo5U•U mRNA wobble interaction, providing a possible rationale for why the ribosome views both as cognate (33).
However, to accommodate the C34•C6 mismatch, the tip of ASL
nucleotide C34 (N4 atom) moves toward the mRNA by 1.6 Å (Fig.
2C). The wobble mRNA C6 position is rigidly held in place,
possibly because of the coordination of a Mg2+ to its 2′-OH and
the base of 16S rRNA residue G530 as seen in other cognate
tRNA–mRNA structures (32) (Fig. 2D). The coordination of the
Mg2+ by both mRNA and 16S rRNA may restrict the movement of
the A-site mRNA, necessitating the ASL conformational changes
that we observe. The length of the codon–anticodon helix is
maintained by 16S rRNA C1054, which stacks beneath C34 of the
ASL, indicating that base stacking is not affected by the C•C
mismatch pairing.
During tRNA selection, three highly conserved 16S rRNA
nucleotides (A1492, A1493, and G530) monitor the codon–anticodon interaction in the A site. These three 16S rRNA nucleotides
directly inspect the minor groove formed by the codon–anticodon
helix to probe for Watson–Crick pairing at the first and second
positions (31). In our structure, despite the near-cognate interaction with a single mismatch at the wobble position, rRNA
interacts identically with the codon–anticodon helix as observed in
other ribosome structures containing cognate tRNA–mRNA pairs
(31, 32) (Fig. S2) and even other near-cognate pairs (34, 35). Additionally, 23S rRNA residue 1913 interacts directly with the 2′-OH
of ASL nucleotide 37, despite the adjacent inserted nucleotide.
A 7-nt anticodon loop is highly conserved across all domains
of life and known to diverge in only rare cases, such as in yeast
mitochondrial tRNAThr (36). The G37.5 insertion leads to an
increase in the minor groove width of the anticodon loop, which
is most pronounced (∼5 Å) immediately adjacent to the insertion
between nucleotides 31–33 (Fig. 3A). This increased minor
groove width leads to an increase in RNA helical twist, which is
distributed over the length of the entire anticodon stem (Fig. 3 A
and B). Although there is a large conformational movement of
the phosphate backbone, base pairing of the stem seems to be
back in standard register by the nucleotides 29–41 pairing (Fig.
3A). On the other side of the anticodon loop, the structural integrity of the conserved U-turn motif is maintained. Because the
phosphate backbone movement of the 5′ stem is distributed
over the length of the ASL, structural superpositioning of ASLSufA6
Maehigashi et al.
base pair. We solved two structures of 70S bound to ASLPro,
which contains a 7-nt anticodon loop and an m1G37 modification, decoding both +1 suppressible codons CCC-G and CCC-U
(Fig. S4 and Table S2). An identical C34•C6 mismatch pair
BIOCHEMISTRY
Fig. 2. Details of the interaction of ASLSufA6 with mRNA in the ribosomal A
site. (A) Watson–Crick base pairs at the first two positions of the A-site codon
(C4 and C5) form when ASLSufA6 decodes a CCC-N codon, whereas a mismatch interaction occurs between ASL C34•C6 codon at the wobble position
(shown with codon G7; codon numbering starts from the P-site codon). The
inserted nucleotide G37.5 is 3′ of the anticodon (red), whereas the nucleotide that lacks any electron density (U32) is shown in blue. (B) A detailed
view of the base pairing between C34 of ASLSufA6 and the C6 or wobble
position of the mRNA. (Upper) ASLSufA6 forms the C•C mismatch pair in the
context of a +1 frameshift CCC-G codon but (Lower) can also form a Watson–
Crick C-G base pair with a cognate CCG codon. 2Fo-Fc electron density
map contoured at 1.0 σ is shown in blue. The overall width of the C34•C6
mismatch and C34–G6 base pair is similar at 10.7 and 11.0 Å, respectively, as
measured between C1′ atoms as depicted by the bar. (C) A comparison of
the position of ASLSufA6 C34 when decoding either a +1 suppressible codon
(purple) or a cognate proline codon (green) shows an ∼1.6 Å movement of
C34 (N4 of the base) to allow the C34•C6 pairing. (D) Direct coordination
between the 2′-OH of C6 of the mRNA, a Mg2+ ion (green sphere), and 16S
rRNA residue G530 (gray) fixes the position of C6 requiring C34 movement to
accommodate the C•C pair. Fo-Fc difference electron density maps are contoured at 3 σ (green mesh), indicating the presence of the Mg2+ ion.
onto ASLPro reveals little variation in its global position (Fig. 3A
and Fig. S3).
ASLSufA6 Undergoes a Context-Dependent Conformational Change at
U32. Important tertiary features govern ASL stability, including
a hydrogen bond between the O2 of a pyrimidine at position 32
and the N6 of A38 (Fig. 3 B and C). The insertion of G37.5 in
ASLSufA6 impedes the formation of the U32–A38 interaction,
because G37.5 occupies the equivalent physical position of A38
in the ASL (Fig. 3A). Despite excellent electron density for most
of the defining features of the ASL in all three structures of 70SASLSufA6 decoding +1 suppressible codons, there is a distinct
absence of electron density for U32, which is located on the
opposite side of the loop from the G37.5 insertion (Fig. 4A).
To determine if this disordering of U32 is an inherent property of
ASLSufA6, we solved a structure of 70S ribosome-ASLSufA6 decoding a cognate proline codon (CCG) (Fig. 4B and Table S1).
Surprisingly, in this mRNA–ASL context, the electron density for
U32 is clearly visible (Fig. 4B). The presence of U32 electron
density in this context indicates that disordering of U32 is not inherently caused by the G37.5 insertion in tRNASufA6. Rather, it is
the combination of the suppressor tRNA and mRNA sequence
context that seems to drive the observed flexibility of U32.
We next examined whether the disordering of U32 is structurally characteristic of the +1 suppressible or C•C near-cognate
Maehigashi et al.
Fig. 3. Remodeling of ASLSufA6 structure induced by the G37.5 insertion. (A)
Overview of the 8-nt anticodon loop of ASLSufA6 (green) compared with the
canonical 7-nt anticodon loop of ASLPro (gray). The G37.5 insertion (red)
causes a widening of the ASL minor groove by ∼5 Å between nucleotides 30
and 32 located on the opposite side of the insertion site. In ASLSufA6, G31 and
U32 span the distance of 3 nt (G37.5, A38, and C39) on the 3′ side of the ASL;
however, in canonical tRNAs, such as tRNAPro, G31 and U32 span only 2 nt
(A38 and C39) on the 3′ side of the ASL. In both cases, the G31–C39 base pair
forms the boundary of the base-paired stem region. The ribose and base of
ASLSufA6 nucleotide U32 (blue) are not observed in electron density maps,
although its phosphate and the following phosphate are visible, allowing its
position to be approximated. (B) Secondary representations of both ASLPro
and ASLSufA6 show that expansion of the ASLSufA6 anticodon loop to 8 nt
abrogates the conserved hydrogen bond between U32 and A38 (boxed) and
alters the incline of base pairs in ASLSufA6. (C) The U32–A38 interaction in
ASLPro (gray) shown alongside U32 (blue) and G37.5 (red) of ASLSufA6, emphasizing the lack of interaction.
PNAS Early Edition | 3 of 6
the loss of the m1G modification at position 37 disrupts the U32–A38
interaction in a manner similar to when ASLSufA6 decodes +1
suppressible codons (Fig. 4A). The structural similarities in
modification-deficient ASLPro and suppressor ASLSufA6 suggest
that they share a common mechanism to induce +1 frameshifting.
Fig. 4. Dynamics of ASL nucleotide U32 are controlled by the ASL and
mRNA context. The ASLSufA6 G37.5 insertion (red) interferes with the U32–
A38 base pair, increasing the dynamics of U32 (blue) such that it undergoes
an ordered to disordered conformational switch depending on whether it is
bound to (A) a +1 frameshifting codon CCC-A/G/U or (B) a cognate CCG
codon. Anticodon nucleotides 34, 35, and 36 that interact with the mRNA
codon are indicated by an arc, whereas U32 is indicated with an arrow. (C)
The absence or (D) presence of the m1G modification at G37 in ASLPro that is
also known to promote +1 frameshifting controls the dynamics of the 5′
stem of the ASL when decoding a cognate CCG codon. 2Fo-Fc electron
density contoured at 1.0 σ is shown in blue.
forms at the wobble position while electron density for U32 is
observed (Fig. S5). These results indicate that U32 disordering is
the consequence of a specific context: the noncanonical 8-nt
anticodon loop of ASLSufA6 binding to a suppressible codon with
the pattern CCC-N within the ribosomal A site.
Modification State of G37 in ASLPro Also Controls U32 Dynamics.
RNA modifications of tRNAs are widespread, and ASL modifications are known to influence decoding (37). Both the anticodon nucleotides 34 and 37 are highly modified, and the lack
of modifications increases frameshifting (38, 39). Modifications
at the ASL wobble nucleotide 34 mainly facilitate unusual base
pairing to expand the genetic code (33, 37, 40), whereas nucleotide 37 modifications participate in stacking interactions with
other anticodon nucleotides and may preorder anticodon nucleotide 36 for mRNA recognition (41, 42). Specifically, the lack
of m1G37 modification in tRNAPro promotes +1 frameshifting
on near-cognate codons (43, 44). The potential mechanistic similarities of this phenomenon to ASLSufA6-mediated frameshifting led
us to investigate how the m1G37 modification of tRNAPro affects its
structure at the decoding center. We first solved the structure of
ASLProm1G37 decoding its cognate CCG codon bound to the
70S ribosome (Fig. 4D). This complex adopts a conformation
consistent with the ribosome recognizing a cognate codon–anticodon pair (31, 32). Next, we determined the structure of 70S
bound to ASLPro lacking the m1G37 modification with the same
CCG codon (Fig. 4C). In this structure, nucleotides 30–32 of the ASL
have poor electron density compared with the 70S-ASLProm1G37
structure (Fig. 4 C and D). This lack of electron density suggests that
4 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1409436111
Discussion
The work here provides a structural basis for the roles of tRNA
structure, mRNA sequence, and RNA modifications in the
noncanonical reading of the genetic code. Although ASLSufA6
contains an additional nucleotide insertion within the anticodon
loop, this extra nucleotide does not cause a rearrangement of the
anticodon that allows for a 4-nt interaction between the codon
and anticodon. Instead, an ASL C34•C6 mismatch forms at
the wobble position upon ASLSufA6 decoding one of the +1
suppressible proline codons: CCC-A, CCC-G, or CCC-U (30).
The overall geometry of the C34•C6 pairing is nearly superimposable with modified wobble base interactions as seen in 30S
structures containing an ASL cmo5U34 decoding either C or U
(33). Both our results and the 30S-cmo5U34•U/C structures
show movements of the anticodon nucleotides toward the
mRNA to accommodate these unusual pairings, suggesting
a mechanism by which the wobble position achieves plasticity. In
the case of ASLSufA6, the phosphate backbone on the opposite
strand to the G37.5 insertion undergoes a conformational rearrangement that considerably widens the minor groove of the
ASL by a maximum of 5 Å (Fig. 3A). In contrast, with the 70Scmo5U•U/C pairings, the anticodon only shifts closer to the
codon to facilitate this pairing, and no additional movements are
noted elsewhere in the ASL (33).
Modifications 3′ to the anticodon at tRNA position 37 are
found on the same isoacceptor tRNAs throughout all three
kingdoms, implying their evolutionary importance (45). Deletion
of the methyltransferase that modifies nucleotide 37 in tRNAProGGG
causes substantial increases in +1 frameshifting (43, 44). Our
structures of ASLPro decoding its cognate codon showed striking
differences depending on the presence of the m1G37 modification (Fig. 4 C and D). Without the modification, U32 is presumed to be conformationally dynamic because of the lack of
electron density that we observe (Fig. 4D), similar to the 70SASLSufA6 structures where the disorder of U32 correlates with
this suppressor decoding +1 suppressible codons CCC-A/G/U
(disordered) or a cognate CCC codon (ordered) (Fig. 4 A and
B). In support of the importance of U32 in maintaining the integrity of the ASL are previous 30S structural studies of engineered ASLs containing an extra nucleotide 5′ of the anticodon
that also cause a +1 reading of specific mRNA codons (46).
Structures of these ASLs revealed a similar destabilization of the
5′ anticodon stem, also disrupting the U32–A38 pair (46).
The structural changes that we observe in ASLSufA6 are distinct from another suppressor tRNA, the Hirsh suppressor. The
Hirsh suppressor tRNA mediates UGA stop codon readthrough
by decoding a C34•A6 mismatch pairing at the wobble position
facilitated by a distortion in the tRNA body caused by a G24A
mutation (34, 47). A comprehensive tRNA distortion seen with
both A9C and G24A mutations extends to both extreme ends of
the tRNA (34): to the decoding center to promote stop codon
readthrough and to the GTPase center, where an increase in the
rate of GTP hydrolysis by EF-Tu occurs during tRNA selection
(48). In contrast, although the ASLSufA6 G37.5 insertion in the
anticodon loop affects the structure of the base-paired stem, we
predict that structural perturbations are not propagated along
the entire tRNA body, because the ASL is almost entirely back
into register by the 28–41 bp (Fig. 3A).
Instead, what seems to promote +1 decoding by ASLSufA6 is
the lack of a 32–38 interaction, which has previously been shown
to be important for correct tRNA selection (49, 50). The nucleotide identity of the 32–38 pair is finely tuned to both the
Maehigashi et al.
mRNA is in the +1 frame based on toeprint primer extension
assays of suppressor tRNAs (54, 55), it is unclear precisely how
and where the +1 frameshift event occurred.
Based on decades of genetic, biochemical, and structural
studies, two likely possibilities exist to explain the molecular basis
of +1 frameshifting (11). The first possibility is an alteration in
translocation of the tRNA–mRNA pair on the 30S from the A to
P site by translation factor EF-G (Fig. 5B). The second possibility
is that after canonical 3-nt translocation, the ribosome loses its
grip on the P-site tRNA stem and slips in the +1 direction. Although
our structures here are of +1 frameshifting-prone ASLs in the A
site, there are hints as to how +1 frameshifting occurs in this particular context. The loss of the U32–A38 base pair in two +1 frameshifting contexts, modification-deficient ASLPro and ASLSufA6,
implies that the integrity of this base pair plays a significant role in
+1 frameshifting (Fig. 5A). In support of this concept, the U32–
A38 base pair has been shown to dramatically affect tRNA discrimination of cognate vs. near-cognate codons both in vitro and in
vivo, demonstrating an important role in stability and function (49,
50). Although the ribosome does not directly contact U32–A38,
the destabilization of the stem caused by the removal of the U32–
A38 interaction may be recognized by EF-G during translocation
(Fig. 5B). Evidence for this comes from a recent cryo-EM pretranslocation structure of 70S bound to EF-G, where domain IV
residues appear to interact with the 5′ stem of the ASL proximal to
U32 (56). The process of translocation involves the dynamic
remodeling of intersubunit bridge interactions and the ∼20-Å relocation of the mRNA–tRNA pair. Therefore, it is tempting to
hypothesize that EF-G causes a rearrangement of the anticodon
loop by virtue of its interactions with the ASL stem, and then, direct
gripping with 16S rRNA and S9 residues in the P site facilitates
conformational rearrangements of the stem. The remodeling of
the P-site tRNA results in changes that are propagated to the
codon–anticodon interaction, allowing a readjustment into the +1
frame. These data are consistent with and provide a new molecular basis for previously proposed repairing models, whereby
mRNA–tRNA repairing is initiated by the ribosome gripping the
ASL stem (30).
Although it is unlikely that a single mechanism exists to explain all occurrences of mRNA frameshifting (11, 57), this study
represents a first step toward understanding the molecular details
required for +1 frameshifting. We envisage that most +1 frameshift suppressor tRNAs adopt noncanonical conformations in the
5′ region of the ASL that mediate a rearrangement in the P site.
Materials and Methods
Ribosome Purification and Crystallization. All mRNAs and unmodified ASLs
were chemically synthesized and purchased from IDT (Table S3), m1G modified ASLs were purchased from Dharmacon, and E. coli tRNAfMet was purchased from Chemical Block Ltd. Purification of T. thermophilus HB8 70S
ribosomes, the formation of 70S complexes, crystallization trials, and
cryoprotection are as previously described (32).
Fig. 5. A model for +1 frameshifting for tRNASufA6 and m1G37-deficient
tRNAPro. (A) In the A site, the ASL (gray) interacts with a 3-nt codon (yellow)
with the additional nucleotide (7) that is read as the preceding codon shown
in red. The conserved 32–38 are depicted as blue and green, respectively. The
anticodon nucleotides 34–36 and mRNA codons 4–6 are closely monitored by
16S rRNA residues G530, C1054, A1492, and A1493 (light purple) and 23S
rRNA A1913 (yellow). The architecture of the A site implies that a 3-nt
codon–anticodon interaction is exclusively read by the ribosome. (B) Domain IV
of EF-G (teal) interacts with the ASL 5′ stem and nucleotide 32, potentially
inspecting the integrity of the anticodon loop. (C) On movement into the P
site by EF-G, the 16S rRNA residues A1338, G1339 (light purple), S9 (purple),
and S13 (pink) inspect the stem of the ASL, whereas very few interactions are
made with the codon–anticodon helix, which is now in a +1 frame.
Maehigashi et al.
Structural Studies of 70S Ribosome Complexes. X-ray diffraction data were
collected at either the Southeast Regional Collaborative Access Team 22-ID
beamline or the Northeastern Collaborative Access Team ID24-C or ID24-E
beamlines at the Advanced Photon Source. Data were integrated and scaled
using the program XDS (58), and solved by molecular replacement using
coordinates from a 70S structure containing mRNA and tRNAs (Tables S1 and
S2) (Protein Data Bank ID codes 3I9D and 3I9E) (59). Crystallographic refinement was performed using the PHENIX software suite (60) followed by
iterative rounds of manual building in Coot (61). Additional refinement
details can be found in SI Materials and Methods. All figures were prepared
in PyMOL (www.pymol.org).
Structural Comparisons. Comparisons of RNA helical parameters for ASLSufA6
bound to the CCC-U codon vs. ASLPro bound to the CCC-U codon were made
using the program 3DNA (62). The modeling of ASLSufA6 in the ribosomal
P site was done by superpositioning the A-site ASLSufA6 model onto the P-site
tRNAfMet using the least-squares fit in Coot (61).
PNAS Early Edition | 5 of 6
BIOCHEMISTRY
strength of the codon–anticodon interaction and the corresponding
aminoacyl group to ensure uniform binding of tRNAs to the ribosome. Any changes to this pair increase near-cognate tRNA
incorporation (49, 50). Our structural results, along with structural studies of 30S-extended ASL complexes (46), imply that
insertions that perturb the structural integrity of the anticodon
loop of suppressor tRNAs may direct recoding by destabilization
of the 32–38 pairing.
Many models, including both the quadruplet and yardstick
models, argued for shifting of the mRNA frame during decoding
in the A site of the ribosome (25). However, structural studies of
the ribosome showed close monitoring of the codon–anticodon
helix and the anticodon loop by rRNA, reflecting the importance
of mRNA frame maintenance in the A site (32). Conversely,
there are no ribosomal interactions with the ASL stem. Based on
our results and other structural studies of the ribosome (32, 51),
we propose that, by virtue of the space and size restrictions in the
A site, the ribosome exclusively decodes three nucleotides in the A
site (Fig. 5A). This three-nucleotide decoding occurs regardless of
whether there is an insertion in the anticodon loop, the absence of
tRNA modifications, or the propensity to near-cognate base pair.
Most, if not all, frameshifting models that depict a larger than
3-nt interaction between the codon and the anticodon in the A
site are incompatible with our structural understanding of the
decoding center.
Upon tRNA–mRNA translocation to the P site, the interactions between the tRNA and the ribosome entirely readjust to
allow strict inspection of the anticodon stem with minimal contact with the codon–anticodon helix (Fig. 5C) (32). The P-site
tRNA is held rigidly in place by 16S rRNA residues A1338 and
G1339 and ribosomal proteins S9 and S13 to properly orient the
tRNA for peptide bond formation. Additionally, 16S rRNA
G1338, A1339, and A790 are proposed to function as a gate
between the P and E sites (32, 52), but they may also inspect the
structural integrity of the P-site tRNA. Upon superpositioning of
ASLSufA6 into the P site, the 16S rRNA residues and the Cterminal tail of S9 interact with the ASL stem directly adjacent to
the U32–A38 pair; these interactions may alter the dynamics of
U32 (Fig. 5C). Indeed, mutation of the terminal residue of S9
alone (Arg128 in Escherichia coli and Arg130 in T. thermophilus)
has been shown to promote +1 frameshifting, implicating its
important role in frame maintenance (53). Although it is clear
that after the mRNA–tRNA pair is translocated to the P site, the
ACKNOWLEDGMENTS. We thank Frank M. Murphy IV and staff members of
the NE-CAT beamlines for assistance during data collection and Graeme
L. Conn, Crystal E. Fagan, and Marc A. Schureck for critical reading of the
manuscript. Research reported in this publication was supported by National
Institute of General Medical Sciences of the National Institutes of Health
Grant R01GM093278 (to C.M.D.). This work is based on research conducted
at the Advanced Photon Source on the Northeastern Collaborative Access
Team beamlines, which is supported by National Center for Research Resources
National Institutes of Health Grant RR-15301, and the Southeast Regional
Collaborative Access Team beamline. Use of the Advanced Photon Source, an
Office of Science User Facility operated for the US Department of Energy Office
of Science by the Argonne National Laboratory, was supported by US
Department of Energy Contract DE-AC02-06CH11357. C.M.D. is a Pew Scholar
in the Biomedical Sciences.
1. Bouadloun F, Donner D, Kurland CG (1983) Codon-specific missense errors in vivo.
EMBO J 2(8):1351–1356.
2. Edelmann P, Gallant J (1977) Mistranslation in E. coli. Cell 10(1):131–137.
3. Jørgensen F, Kurland CG (1990) Processivity errors of gene expression in Escherichia
coli. J Mol Biol 215(4):511–521.
4. Kurland CG (1992) Translational accuracy and the fitness of bacteria. Annu Rev Genet
26:29–50.
5. Zaher HS, Green R (2009) Quality control by the ribosome following peptide bond
formation. Nature 457(7226):161–166.
6. Riddle DL, Roth JR (1970) Suppressors of frameshift mutations in Salmonella typhimurium. J Mol Biol 54(1):131–144.
7. Riyasaty S, Atkins JF (1968) External suppression of a frameshift mutant in salmonella.
J Mol Biol 34(3):541–557.
8. Yourno J, Tanemura S (1970) Restoration of in-phase translation by an unlinked
suppressor of a frameshift mutation in Salmonella typhimurium. Nature 225(5231):
422–426.
9. Riddle DL, Roth JR (1972) Frameshift suppressors. II. Genetic mapping and dominance
studies. J Mol Biol 66(3):483–493.
10. Kohno T, Roth JR (1978) A Salmonella frameshift suppressor that acts at runs of A
residues in the messenger RNA. J Mol Biol 126(1):37–52.
11. Atkins JF, Björk GR (2009) A gripping tale of ribosomal frameshifting: Extragenic
suppressors of frameshift mutations spotlight P-site realignment. Microbiol Mol Biol
Rev 73(1):178–210.
12. Riddle DL, Carbon J (1973) Frameshift suppression: A nucleotide addition in the anticodon of a glycine transfer RNA. Nat New Biol 242(121):230–234.
13. Roth JR (1981) Frameshift suppression. Cell 24(3):601–602.
14. Magliery TJ, Anderson JC, Schultz PG (2001) Expanding the genetic code: Selection of
efficient suppressors of four-base codons and identification of “shifty” four-base
codons with a library approach in Escherichia coli. J Mol Biol 307(3):755–769.
15. Riddle DL, Roth JR (1972) Frameshift suppressors. 3. Effects of suppressor mutations
on transfer RNA. J Mol Biol 66(3):495–506.
16. Yourno J (1972) Externally suppressible +1 “glycine” frameshift: Possible quadruplet
isomers for glycine and proline. Nat New Biol 239(94):219–221.
17. Sroga GE, Nemoto F, Kuchino Y, Björk GR (1992) Insertion (sufB) in the anticodon loop
or base substitution (sufC) in the anticodon stem of tRNA(Pro)2 from Salmonella typhimurium induces suppression of frameshift mutations. Nucleic Acids Res 20(13):
3463–3469.
18. Cummins CM, Donahue TF, Culbertson MR (1982) Nucleotide sequence of the SUF2
frameshift suppressor gene of Saccharomyces cerevisiae. Proc Natl Acad Sci USA
79(11):3565–3569.
19. Gaber RF, Culbertson MR (1984) Codon recognition during frameshift suppression in
Saccharomyces cerevisiae. Mol Cell Biol 4(10):2052–2061.
20. Spirin AS (1986) Ribosome Structure and Protein Biosynthesis (Benjamin/Cummings
Publishing Company, Inc., Menlo Park, CA).
21. Bossi L, Roth JR (1981) Four-base codons ACCA, ACCU and ACCC are recognized by
frameshift suppressor sufJ. Cell 25(2):489–496.
22. Bossi L, Smith DM (1984) Suppressor sufJ: A novel type of tRNA mutant that induces
translational frameshifting. Proc Natl Acad Sci USA 81(19):6105–6109.
23. Moore B, Persson BC, Nelson CC, Gesteland RF, Atkins JF (2000) Quadruplet codons:
Implications for code expansion and the specification of translation step size. J Mol
Biol 298(2):195–209.
24. Curran JF, Yarus M (1987) Reading frame selection and transfer RNA anticodon loop
stacking. Science 238(4833):1545–1550.
25. Anderson JC, Magliery TJ, Schultz PG (2002) Exploring the limits of codon and anticodon size. Chem Biol 9(2):237–244.
26. Gaber RF, Culbertson MR (1982) The yeast frameshift suppressor gene SUF16-1 encodes an altered glycine tRNA containing the four-base anticodon 3′-CCCG-5′. Gene
19(2):163–172.
27. Atkins JF, Gesteland RF, Reid BR, Anderson CW (1979) Normal tRNAs promote ribosomal frameshifting. Cell 18(4):1119–1131.
28. Tucker SD, Murgola EJ, Pagel FT (1989) Missense and nonsense suppressors can correct
frameshift mutations. Biochimie 71(6):729–739.
29. Schmeing TM, Ramakrishnan V (2009) What recent ribosome structures have revealed
about the mechanism of translation. Nature 461(7268):1234–1242.
30. Qian Q, et al. (1998) A new model for phenotypic suppression of frameshift mutations
by mutant tRNAs. Mol Cell 1(4):471–482.
31. Ogle JM, et al. (2001) Recognition of cognate transfer RNA by the 30S ribosomal
subunit. Science 292(5518):897–902.
32. Selmer M, et al. (2006) Structure of the 70S ribosome complexed with mRNA and
tRNA. Science 313(5795):1935–1942.
33. Weixlbaumer A, et al. (2007) Mechanism for expanding the decoding capacity of
transfer RNAs by modification of uridines. Nat Struct Mol Biol 14(6):498–502.
34. Schmeing TM, Voorhees RM, Kelley AC, Ramakrishnan V (2011) How mutations in
tRNA distant from the anticodon affect the fidelity of decoding. Nat Struct Mol Biol
18(4):432–436.
35. Demeshkina N, Jenner L, Westhof E, Yusupov M, Yusupova G (2012) A new understanding
of the decoding principle on the ribosome. Nature 484(7393):256–259.
36. Li M, Tzagoloff A (1979) Assembly of the mitochondrial membrane system: Sequences
of yeast mitochondrial valine and an unusual threonine tRNA gene. Cell 18(1):47–53.
37. Agris PF, Vendeix FA, Graham WD (2007) tRNA’s wobble decoding of the genome: 40
years of modification. J Mol Biol 366(1):1–13.
38. Urbonavicius J, Qian Q, Durand JM, Hagervall TG, Björk GR (2001) Improvement of
reading frame maintenance is a common function for several tRNA modifications.
EMBO J 20(17):4863–4873.
39. Bouadloun F, Srichaiyo T, Isaksson LA, Björk GR (1986) Influence of modification next
to the anticodon in tRNA on codon context sensitivity of translational suppression
and accuracy. J Bacteriol 166(3):1022–1027.
40. Nasvall SJ, Chen P, Bjork GR (2004) The modified wobble nucleoside uridine-5oxyacetic acid in tRNAPro(cmo5UGG) promotes reading of all four proline codons in
vivo. RNA 10(10):1662–1673.
41. Grosjean H, Söll DG, Crothers DM (1976) Studies of the complex between transfer
RNAs with complementary anticodons. I. Origins of enhanced affinity between
complementary triplets. J Mol Biol 103(3):499–519.
42. Dao V, et al. (1994) Ribosome binding of DNA analogs of tRNA requires base modifications and supports the “extended anticodon” Proc Natl Acad Sci USA 91(6):
2125–2129.
43. Björk GR, Wikström PM, Byström AS (1989) Prevention of translational frameshifting
by the modified nucleoside 1-methylguanosine. Science 244(4907):986–989.
44. Hagervall TG, Tuohy TM, Atkins JF, Björk GR (1993) Deficiency of 1-methylguanosine
in tRNA from Salmonella typhimurium induces frameshifting by quadruplet translocation. J Mol Biol 232(3):756–765.
45. Björk GR (1995) Genetic dissection of synthesis and function of modified nucleosides
in bacterial transfer RNA. Prog Nucleic Acid Res Mol Biol 50:263–338.
46. Dunham CM, et al. (2007) Structures of tRNAs with an expanded anticodon loop in
the decoding center of the 30S ribosomal subunit. RNA 13(6):817–823.
47. Hirsh D (1970) Tryptophan tRNA of Escherichia coli. Nature 228(5266):57.
48. Cochella L, Green R (2005) An active role for tRNA in decoding beyond codon:anticodon pairing. Science 308(5725):1178–1180.
49. Ledoux S, Olejniczak M, Uhlenbeck OC (2009) A sequence element that tunes
Escherichia coli tRNA(Ala)(GGC) to ensure accurate decoding. Nat Struct Mol Biol
16(4):359–364.
50. Olejniczak M, Uhlenbeck OC (2006) tRNA residues that have coevolved with their anticodon to ensure uniform and accurate codon recognition. Biochimie 88(8):943–950.
51. Jenner L, Demeshkina N, Yusupova G, Yusupov M (2010) Structural rearrangements
of the ribosome at the tRNA proofreading step. Nat Struct Mol Biol 17(9):1072–1078.
52. Schuwirth BS, et al. (2005) Structures of the bacterial ribosome at 3.5 A resolution.
Science 310(5749):827–834.
53. Näsvall SJ, Nilsson K, Björk GR (2009) The ribosomal grip of the peptidyl-tRNA is
critical for reading frame maintenance. J Mol Biol 385(2):350–367.
54. Phelps SS, et al. (2006) Translocation of a tRNA with an extended anticodon through
the ribosome. J Mol Biol 360(3):610–622.
55. Walker SE, Fredrick K (2006) Recognition and positioning of mRNA in the ribosome by
tRNAs with expanded anticodons. J Mol Biol 360(3):599–609.
56. Brilot AF, Korostelev AA, Ermolenko DN, Grigorieff N (2013) Structure of the ribosome with elongation factor G trapped in the pretranslocation state. Proc Natl Acad
Sci USA 110(52):20994–20999.
57. Dinman JD (2012) Mechanisms and implications of programmed translational frameshifting. Wiley Interdiscip Rev RNA 3(5):661–673.
58. Kabsch W (2010) Xds. Acta Crystallogr D Biol Crystallogr 66(Pt 2):125–132.
59. Jenner LB, Demeshkina N, Yusupova G, Yusupov M (2010) Structural aspects of
messenger RNA reading frame maintenance by the ribosome. Nat Struct Mol Biol
17(5):555–560.
60. Adams PD, et al. (2010) PHENIX: A comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr 66(Pt 2):213–221.
61. Emsley P, Lohkamp B, Scott WG, Cowtan K (2010) Features and development of Coot.
Acta Crystallogr D Biol Crystallogr 66(Pt 4):486–501.
62. Lu XJ, Olson WK (2003) 3DNA: A software package for the analysis, rebuilding and
visualization of three-dimensional nucleic acid structures. Nucleic Acids Res 31(17):
5108–5121.
6 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1409436111
Maehigashi et al.