J. Mol. Biol. (1997) 266, 877±890
Analysis of Interactions Between the Codon ±
Anticodon Duplexes within the Ribosome: Their Role
in Translation
Valery I. Lim
Institute of Protein Research
Russian Academy of Sciences
142292, Pushchino, Moscow
Region, Russia
Computer graphics simulation of interactions between the codon ±anticodon duplexes formed by normal elongator tRNAs at the ribosomal A, P
and E-sites (the AP and PE interduplex interactions) was made. This
demonstrated that only the correct duplexes at the A-site are compatible
with the AP interduplex interaction. The selection of synonymous codons
and anticodon wobble bases, together with the AP interduplex interaction, prevents frameshifting. In the absence of this interaction the ef®ciency of the selection falls off sharply. This suggests that the AP
interduplex interaction should be retained during translocation and in
the post-translocation state, i.e. the PE interduplex interaction that is
identical with that of AP should exist to avoid frameshifting. In such a
model the P-site duplex provides an indirect linkage between the A and
E-site duplexes. The indirect linkage prohibits the simultaneous existence
of the A and E-site duplexes. The wobble pairs of the P and E-site
duplexes can affect the rate of the A-site occupation via the AP interduplex interaction and the AE interduplex indirect linkage. It is demonstrated that frameshifting can occur from the AP or PE codon± anticodon
complex destabilization caused, for example, by small mobility of the
wobble pairs, misreading of the codon, unmodi®ed adenine and guanine
at tRNA positions 34 (wobble) and 37, respectively. The results obtained
can be subjected to direct experimental tests.
# 1997 Academic Press Limited
Keywords: codon-anticodon interaction; codon context effect; translation;
misreading; frameshifting
Introduction
The rate at which a codon is read appears not to
be constant but depends on the identity of the codon's neighbours, a phenomenon known as the
context effect (for reviews, see Murgola, 1990;
Hat®eld & Gutman, 1992; Osawa et al., 1992; Yarus
& Curran, 1992; Buckingham, 1994). One possible
source of the context effect is interactions between
the codon± anticodon duplexes and anticodon
loops. Genetic, physical and theoretical studies
suggest that the wobble base-pair of the codon ±anticodon duplex located at the ribosomal P (peptidyl-tRNA) site may interact with the A
(aminoacyl-tRNA) site duplex (Lim et al., 1992;
Yarus & Smith, 1995). Such an interaction (the AP
interduplex interaction; Figure 1) can be observed
at different mutual orientations of the tRNAs, since
signi®cant changes in the mutual orientation
0022±2836/97/100877±14 $25.00/0/mb960802
caused by sterically allowed conformational
changes in a tRNA can occur without disturbance
of the AP interduplex interaction (see below, property 5 of the AP interduplex interaction). The
characteristic feature of the AP interduplex interaction is that the wobble base-pair of the P-site
codon ± anticodon duplex is stacked on the sugarphosphate moiety of the A-site codon and that this
stacking interaction shields the two A-site codon
inter-ribose hydrogen bonds (Figure 1(b)).
Direct localisation of tRNAs within the elongating
ribosome has been attempted using a newly developed neutron-scattering technique, the nuclear spin
contrast variation (Nierhaus et al., 1995). This
physical study has revealed that the A and P-site
tRNAs are separated by the dihedral angle of 50
formed by their planes, and that the mutual arrangement of these tRNAs does not change during
translocation. Very recently a re®ned value (100 )
# 1997 Academic Press Limited
878
of this dihedral angle has been obtained (Dr K. H.
Nierhaus, personal communication) using the latest ``Frank model'' of the ribosome (Frank et al.,
1995). This value of the dihedral angle is in complete agreement with that found theoretically
(100(10) ; Lim & Spirin, 1986; Spirin & Lim,
1986; Lim & Venclovas, 1992). At these values of
the dihedral angle, the AP interduplex interaction
is observed virtually without distortion of the
crystal structure of the tRNAs (Figure 1).
The Ap tRNA-mRNA-tRNA complex within the
ribosome has also recently been visualized with
cryoelectron microscopy by Agrawal et al. (1996)
and by Dr R. Brimacombe, (personal communication). A cryoelectron microscopy study of
Agrawal et al. (1996) has revealed that the arrangement of the A and P-site tRNAs involves
the dihedral angle of ÿ160 (a re®ned value is
about ÿ110 ; Dr J. Frank, personal communication). With regard to the cryoelectron microscopy data of van Heel's group, they
indicate the dihedral angle of ÿ50 . At these
values (ÿ110 and ÿ50 ) of the dihedral angle,
the AP interduplex interaction cannot be observed without distortion of the crystal structure
of the tRNAs.
Analysis of interactions between the codon ± anticodon duplexes should not be limited to a consideration of the AP interduplex interaction alone, since
besides the traditional A and P tRNA binding
sites, the T (elongation factor Tu) and E (exit) sites,
and the intermediary (hybrid) state A/T, A/P and
P/E have been de®ned (for reviews, see Noller,
1991; Weijland & Parmeggiany, 1994; Yarus &
Smith, 1995). The T-site, or a related formulation
called the R-site (Lake, 1977), is traversed before
the A and P-sites; it is the location of the newly
bound aminoacyl-tRNA when it is still held by
elongation factor Tu (EF-Tu). The E-site was proposed by Wettstein & Noll (1965). It has been
shown (Rheinberger, 1991; Nierhaus, 1993) that
deacylated tRNA does not leave the ribosome immediately from the P-site, but co-translocates from
the P-site to the E-site when peptidyl-tRNA translocates from the A-site to the P-site. At the P and
E-sites, the tRNAs interact with codons and the
deacylated tRNA is expelled from the E-site by the
binding of aminoacyl-tRNA to the A-site at the beginning of the subsequent elongation cycle. Here,
we analyse the in¯uence of the interduplex interactions on ribosomal translation within the framework of the model proposing the AP and PE
interduplex interactions.
Results and Discussion
Allowed codon ± anticodon duplexes: a wobble
in the pairing of the first, second and third
codon bases
All codon± anticodon duplexes fall into two general groups, one with and the other without disallowed steric overlaps and an uncompensated loss
Interactions Between Codon ±Anticodon Duplexes
of hydrogen and polar atom ±ion bonds. The duplexes of these groups should be considered as disallowed and allowed, respectively (see Materials
and Methods).
Our stereochemical analysis (the results will be
published elsewhere) has revealed that all non-canonical base-pairs (the pairs UU, UC, UG, CC, CA,
AA AG and GG) can be incorporated into
codon ± anticodon duplexes of the allowed group.
Non-canonical base-pairs can be formed in any duplex position (even simultaneously in all three positions) using the base-pair propeller twist and
Hoogsteen-like base-pairing (recently Hoogsteenlike trans UU base-pairs have been found in the
crystal structure of an RNA double helix; Wahl
et al., 1996). The propeller twist prevents uncompensated losses of hydrogen bonds in non-canonical base-pairs (Lim, 1994, 1995). Hoogsteen-like
base-pairing permits incorporation into the codonanticodon mini-helix of such base-pairs that, in the
case of Watson-Crick-like base-pairing, are incompatible with a double helix because of uncompensated losses of hydrogen bonds (for example, the
CC and GG pairs). Analysis has demonstrated that
the allowed group is rather large, 20 to 40% of all
conceivable duplexes (642) in accordance with an
assortment of anticodon wobble bases, conserved
tRNA purine base 37 and their modi®cations, since
they affect the formation of the propeller twist in
non-canonical base-pairs of codon± anticodon duplexes.
The available experimental data support a wobble
in the pairing of the ®rst, second and third codon
bases. These data show that there is a wide variety
of non-canonical base-pairs in different regions of
RNA double helices. The non-canonical base-pairs
AG, AC and C are placed at the double helix
ends in the anticodon stems of crystal structures of
yeast tRNAPhe (Kim et al., 1974; Jack et al., 1976;
Stout et al., 1978), yeast tRNAAsp (Moras et al.,
(Woo
1980) and Escherichia coli initiator tRNAMet
f
et al., 1980). These pairs together with canonical
base-pairs of the anticodon stem form a single
double helix. Four consecutive non-canonical basepairs are observed in the middle of the crystal
structure of RNA double helices: GU, CU, UC, UG
(Holbrook et al., 1991; Cruse et al., 1994) and GU,
UU, UU, UG (Baeyens et al., 1995). The UU basepair is observed within a conserved ribosomal
RNA hairpin (Wang et al., 1996) and in the middle
of the anticodon ± anticodon duplexes formed in
the crystals of yeast tRNAAsp (Moras et al., 1980).
Moreover, the difference in the lifetime of anticodon ±anticodon duplexes of a similar type and of
the ``correct'' anticodon ±anticodon duplexes (as
determined by the genetic coding rules) is insuf®cient to avoid translation errors (Grosjean et al.,
1978).
The above-listed experimental and stereochemical
evidence indicates that the codon ± anticodon interaction cannot be explained just by the internal stability of the codon ± anticodon duplexes, since noncanonical pairs are observed in any position of al-
Interactions Between Codon ±Anticodon Duplexes
879
Figure 1. The AP interduplex interaction. (a) Mutual orientation of the ribosomal A and P-site bound tRNAs. The
two CCA ends (black beads) of the tRNAs are drawn together in order to allow formation of the peptide bond, and
the two anticodons form codon ± anticodon mini-helices with adjacent codons (curved tube). Black and hatched trapeziums are the codon ± anticodon base-pairs. The horizontally and vertically hatched trapeziums are the wobble pairs
of the A and P-site codon± anticodon duplexes, respectively. o is the dihedral angle formed by the planes of tRNA
molecules. The long vertical arrow is the axis of the dihedral angle o. The AP interduplex interaction is shown: the
interaction between the A-site codon (the 30 portion of the curved tube) and the wobble pair of the P-site duplex (the
vertically hatched trapezium). (b) Stereo view of the AP interduplex interaction. The structure is drawn in accordance
with the crystallographic coordinates of yeast phenylalanine tRNA at o 100 . In the P-site codon ±anticodn duplex
(right) only the wobble base-pair is shown. The A-site codon triplet and the third residue of the P-site codon are
shown in bold lines. In the A-site anticodon loop (left) the residues 32 to 37 are shown. The anticodon (residues 34 to
36) is paired with the codon (bold lines). Broken lines show the two A-site codon inter-ribose hydrogen bonds and
hydrogen bonding between U33 and phosphate 36 in the A-site anticodon loop. (c) The AP tRNA-mRNA-tRNA complex superimposed on the 70 S ribosome model proposed by Frank et al. (1991). In this model, the CCA ends of the
tRNA molecules are ®xed at the base of the central protuberance (the peptidyl transferase center) of the 50 S subunit,
and the anticodon loops lie in the neck region (the decoding site) of the 30 S subunit. A detailed description of this
Figure see in Lim et al. (1992).
880
Interactions Between Codon ±Anticodon Duplexes
lowed duplexes and the differences in the stability
of the allowed duplexes cannot play a crucial role
in preventing codon misreading. The energetic
differences are an order of magnitude less than the
energy of a hydrogen bond, which is required to
provide the experimentally observed level of misreading of the codon in vivo (see Materials and
Methods). All these facts indicate that ``outside''
factors must affect the formation of the ribosomal
A-site codon ± anticodon duplex. It is demonstrated
below that the AP interduplex interaction can play
the role of the main outside factor in binding the
A-site tRNA.
Properties of the AP interduplex interaction
Property 1. The AP interduplex interaction
constrains the A-site codon in the rigidly fixed
A-form conformation
The inter-ribose hydrogen bonds 20 OH ± O40 are observed in RNA double helices (Kim et al., 1974;
Robertus et al., 1974; Stout et al., 1978; Moras et al.,
1980; Dock-Bregeon et al., 1989). These hydrogen
bonds ®x A-helix geometry. The disruption of the
A-site codon inter-ribose hydrogen bonds in the
AP codon ±anticodon complex should result in a
large energetic loss (20 or 40 kJ/mol, the energy of
one or two normal hydrogen bonds). This proceeds
from shielding of the inter-ribose hydrogen bonds
by the P-site wobble base-pair (Figure 1(b)). The
shielded ribose 20 OH groups and O40 atoms cannot
form polar atom ± ion bonds or new hydrogen
bonds with any outside partners, including solvent
molecules, in exchange for the disrupted inter-ribose hydrogen bonds. That is why the A-site
codon should be ®xed in the A-form by the AP interduplex interaction.
Property 2. Only correct duplexes at the A-site are
compatible with the AP interduplex interaction
Maintenance of the codon in the A-form (property
1) and the structure of the anticodon loop counteract the non-canonical base-pairing in the ®rst and
second positions of the A-site codon (Lim &
Venclovas, 1992) and enable the formation of only
the correct wobble base-pairs at the third position
(Lim, 1994, 1995), i.e. only the correct duplexes at
the A-site are compatible with the AP interduplex
interaction. The formation of the wrong duplexes
(including duplexes formed by anticodons and
near-cognate codons) in the presence of the AP interduplex interaction is accompanied by an energetic loss of 20 to 40 kJ/mol (an energetic
difference between the correct and wrong duplexes
that is required to provide the experimentally observed level of misreading of the codon in vivo; see
Materials and Methods).
Figure 2. The canonical base-pair UA (bold lines),
demonstrating the base-pair major and minor edges.
These edges in a double helix form its major and minor
grooves. Two extrabold lines are glycosyl bonds. Circles
are the polar atoms and groups of the codon base that
in the minor groove of the A-site codon± anticodon
duplex are positioned close to the major edge of the
P-site wobble pair. Filled circles are the adenine or
guanine nitrogen atom N3 and the oxygen atom O2
of the pyrimidine base U or C (®ne lines). The open
circle is the guanine NH2 group.
Property 3. In contrast to the A-site duplex, a wide
variety of non-canonical base-pairs is admissible
in any position of the P-site duplex, even
simultaneously in all three positions
The AP interduplex interaction does not ®x the
codon in the P-site duplex. Therefore the P-site duplex can be considered as an ordinary allowed one.
Consequently, a wide variety of non-canonical
base-pairs can be observed in any position of the
P-site duplex, even simultaneously in all three positions (see above).
Property 4. The AP interduplex interaction
counteracts the formation of both the correct and
the wrong duplexes at the A-site when the wobble
pair in the P-site duplex or solvent molecules and
massive modifications at its major edge are fixed
In the minor groove of any A-site codon ±anticodon duplex the polar atoms O2, N3 and the NH2
groups of the ®rst and second codon bases are positioned close to the major edge of the P-site wobble pair (the polar atoms, groups and the major
edge are shown in Figure 2). Therefore, the P-site
wobble pair should be mobile within the minihelix. Otherwise, certain of the O2 and N3 atoms,
the NH2 groups of the A-site codon and the major
edge polar atoms of the P-site wobble pair will
form, at best, only severely deformed hydrogen or
polar atom ± ion bonds with ordered solvent molecules, a large number of which is observed (Egli
et al., 1996; Wahl et al., 1996) in the minor groove
of the RNA double helix. The ®xation of solvent
molecules (this takes place in adenine; Lim, 1995)
or massive modi®cations (anticodon wobble base
modi®cations) at the major edge of the P-site wob-
881
Interactions Between Codon ±Anticodon Duplexes
ble base-pairs also leads to the formation of severely deformed hydrogen or polar atom ± ion
bonds regardless of the mobility of the P-site wobble base-pair in the mini-helix. All this means that
®xation of the P-site wobble pair as well as that of
solvent molecules and massive modi®cations at its
major edge should counteract the formation of any
duplex at the A-site via the AP interduplex interaction.
In the case of the P-site wobble pairs UU and UC,
besides the atoms O2 and N3, the guanine NH2
groups and the major edge polar atoms, there are
water molecules that also lose a hydrogen or a
polar atom± ion bond. When the codon residue in
the P-site wobble pairs UU and UC is ®xed in the
A-form conformation, one base-base hydrogen
bond in these pairs should be replaced by a water
bridge (Lim & Venclovas, 1992; Lim, 1994). At the
P-site a bridging water molecule loses a hydrogen
or a polar atom ± ion bond as the result of the Asite codon ± anticodon duplex formation. This proceeds from shielding of a bridging water molecule
by the sugar-phosphate backbone of the A-site
codon. In the absence of ®xation of the P-site wobble pair, a bridging water molecule can be removed and base-pairing in the P-site wobble pairs
UU and UC can occur without a water bridge.
There is genetic evidence suggesting that the small
mobility of the P-site wobble pair causes an occlusion of the A-site. Smith & Yarus (1989) have
shown that the substitution of purines for the conserved pyrimidines at anticodon loop positions 32
and 33 of the P-site tRNA reduces the rate of A-site
occupation and that the effect at position 33 is
greater than that at position 32. Curran (1995) has
examined in vivo decoding properties of Escherichia
coli arginine tRNAICG, which is the only tRNA in
E. coli that reads the arginine codons CGU, CGC
and CGA. It was concluded that of the wobble
pairs IU, IC and IA, the P-site purine-purine wobble pair IA destabilizes the A-site codon ± anticodon
duplex.
The genetic data reported by Smith & Yarus (1989)
and Curran (1995) display one structural generality: in both cases a decrease in the mobility of the
P-site wobble pair takes place. Bases 32 and 33 in
the crystallographic structure of tRNA are located
inside the anticodon loop and parallel with its central part (the backbone section between residues 33
and 35 containing the anticodon wobble residue
34; see Figure 1(b)). Therefore a substitution by the
larger purine bases (especially at position 33)
should result in a stretching of the central part
that, in turn, should cause a decrease in the mobility of any wobble pair in the codon± anticodon
mini-helix. With regard to the purine-purine pair
IA, the distance in this pair between the glycosyl
bonds, in contrast to the purine-pyrimidine pairs
IU and IC, strongly differs from that in the canonical pair (Crick, 1966). Therefore, the mobility of the
wobble pair IA is also small. Thus, the results presented demonstrate that the experimental data reported by Smith & Yarus (1989) and Curran (1995)
can actually be explained by a decrease in the mobility of the P-site wobble pair.
Property 5. Significant changes in the mutual
orientation of the tRNAs can occur without
disturbance of the AP interduplex interaction
Small, sterically allowed conformation changes in a
tRNA (especially in the anticodon stem) can be accompanied by a noticeable rearrangement of the
acceptor stem with respect to the codon ±anticodon
duplex. Let us consider one such example. It is
known (Saenger, 1984) that the RNA double helices display two major, structurally similar conformations, the A-form conformation with 11 base
pairs per helix turn (such helices are observed in
the tRNA crystal structure; Kim et al., 1974;
Robertus et al., 1974; Stout et al., 1978; Moras et al.,
1980) and the A0 -form with 12 pairs per turn. Furthermore, X-ray analysis shows that an RNA
double helix can be kinked at points dividing the
helix into blocks of four to ®ve base-pairs (DockBregeon et al., 1989). The kink angle (divergence
from a straight helix) is about 10 . The structural
changes within the double helices of the tRNA anticodon and acceptor arms caused by such kinks
and the A$A0 transitions do not disturb the ``tertiary'' interactions in the L-shaped tRNA molecules. Even these very small structural variations
(Figure 3) allow alteration of ``crystal'' value of
100 (Figure 1) of the angle o by 20 and an increase in the distance between the tRNA CCA
Ê , leaving the AP interdutermini by at least 20 A
plex interaction unchanged.
Besides the kinks and the A$A0 transitions within
the double helix regions of tRNAs, structural
changes beyond the helices in the vicinity of their
ends can occur without disruption of intramolecular hydrogen bonds. For instance, there can be
a rotation of the anticodon loop in the anticodon
stem around a double helix axis. In this case the
sterically allowed rotation of 20 can be performed without disruption of the anticodon conformation. The A$A0 transitions, the structural
changes at the helix ends within the anticodon
stem and kinks in the acceptor stem of tRNAs
can alter the angle o through a wide range (at
least 100(50) ) retaining the AP interduplex
interaction (Figure 3).
It should be pointed out that these intramolecular
conformational variations in tRNAs may provide
intermediate states A/T, A/P and P/E in the
movement of transfer RNA in the ribosome
(Moazed & Noller, 1989), since the ®xation of the
codon-anticodon duplex on the small subunit
(Figure 1(c)) does not counteract the shifts of the
acceptor end of tRNA on the large subunit and vice
versa the ®xation of the acceptor end does not
counteract the shifts of the duplex (for a discussion
of intramolecular conformational variations in
tRNAs and their functions see also the waggle
theory by Yarus & Smith, 1995).
882
Figure 3. Small structural variations in the AP tRNAmRNA-tRNA complex that can occur without disturbance of the AP interduplex interaction (lower part of the
drawing) and that lead to marked changes of the angle
o and the distance between the tRNA CCA ends (black
beads). The curved tube is the sugar-phosphate backbone of two adjacent mRNA codons. A and P are the
anticodon loops of the ribosomal A and P-site bound
tRNAs. Long ®ne lines are the anticodon and acceptor
stem axes of the tRNAs. Rotations around these axes
and their kinks shift the acceptor stem axes. A displacement of the CCA ends toward the viewer (increasing
the angle o) is shown.
The role of the AP interduplex interaction in
the prevention of ribosomal frameshifting
during a normal elongation cycle
Non-slipping and slipping frameshifting
All conceivable cases of frameshifting can be separated into two categories: non-slipping and slipping
frameshifting (Figure 4). Non-slipping frameshifting can formally be modelled by insertions
(‡frameshifting) and deletions (ÿframeshifting) of
mRNA residues within the AP codon± anticodon
complex (Figure 4(a)). In the case of slipping frameshifting, a disengagement of the tRNA anticodons from an initial codon interaction allowing the
mRNA to slip with respect to the tRNAs ± ribosome
complex takes place. The anticodons may then repair with new codons, so that synthesis continues
downstream (Figure 4(b)). Neither of these types of
frameshifting must take place during a normal
elongation cycle.
The interduplex interaction in the AP codon ±
anticodon complex formed by normal elongator
tRNAs should counteract non-slipping frameshifting
The obvious assumption to adopt is that the A and
P-site codon ± anticodon duplexes formed by normal elongator tRNAs are ®xed at the A and Psites, and are positioned relative to each other in a
universal manner that is independent of the tRNA
species involved. This should counteract non-slipping frameshifting, since in the absence of the
Interactions Between Codon ±Anticodon Duplexes
Figure 4. Frameshifting patterns. (a) Non-slipping frameshifting. Acute angles are the tRNA anticodon loops
paired with the codons. Bold lines show the anticodon
loop of peptidyl-tRNA and the zero frame codon. Fine
lines are the anticodon loop of the deacylated tRNA
paired with mRNA. Both ‡1 frameshifting (left, the
insertion of the mRNA residue into the AP codon-anticodon complex) and ÿ1 frameshifting (right, the deletion of the mRNA residue) are shown. (b) Slipping
frameshifting. Tandem slippage of peptidyl-tRNA and
deacylated tRNA (1) as well as single slippage of peptidyl-tRNA (2) are shown.
shifts of the duplexes relative to each other the insertions and deletions of mRNA residues within
the AP codon ± anticodon complex cannot occur
without the disruption of the duplexes (deletions)
and without shielding of oxygen atoms in the
sugar-phosphate backbone or the disruption of the
inter-ribose hydrogen bonds in the A-site codon
(insertions). Consequently, non-slipping frameshifting in the AP codon ± anticodon complex should be
unfeasible when the complex is formed by normal
elongator tRNAs and can be feasible when the
shifts of the A and P-site duplexes relative to each
other (including the expelling of a duplex from the
P-site) are possible. Such shifts can be induced, for
example, by non-standard structures of the anticodon loop or by speci®c interactions between the A
and P-site tRNAs (for possible mechanisms for
non-slipping frameshifting see, e.g. Weiss, 1984;
Curran & Yarus, 1987; BjoÈrk et al., 1989;
Vimaladithan & Farabaugh, 1994, and references
therein).
Tandem slippage of the A and P-site tRNAs
With regard to slipping frameshifting, only tandem
slippage of the A-site tRNA (newly formed peptidyl-tRNA) and P-site tRNA (deacylated tRNA) can
occur in the AP codon ± anticodon complex formed
by normal elongator tRNAs. Slippage of the A-site
tRNA without slippge of the P-site tRNA is impossible because this variant of slipping frameshifting is the equivalent of non-slipping frameshifting
(Figure 4(a)), which is prohibited. Therefore we can
conclude that only slipping frameshifting resulting
from tandem slippage of the A and P-site tRNAs
Interactions Between Codon ±Anticodon Duplexes
can occur in the AP condon ±anticodon complex
formed by normal elongator tRNAs.
Tandem slippage rule
The A-site tRNA after slippage, according to property 2, should form only correct duplexes, whereas
the P-site tRNA is free to interact with any codon
if this codon and the anticodon are able to form an
allowed duplex (property 3) in which the pair
formed by the ®rst anticodon base is not ®xed
within the mini-helix (property 4).
Elimination of tandem slippage by the selection of
synonymous codons and anticodon wobble bases
Assume that the A-site anticodon in the AP
codon± anticodon complex forms the correct duplex with the ith codon in a coding sequence. According to the tandem slippage rule, the second
and third bases of the A-site anticodon after slippage must form canonical base-pairs with new
codon partners. In the case of ‡1 and ‡2 slippages
the third base of the ith codon should be paired
with the second and third anticodon base, respectively. After ÿ2 and ÿ1 slippages the third base of
the i ÿ 1th codon should be paired with the second
and third anticodon base, respectively. In all these
four cases the selection of the third base in the
i ÿ 1th and ith codons (selection of synonymous
codons) can prevent the canonical base-pairing at
the second or third anticodon positions, i.e. slippage of the A-site anticodon in the AP codon ± anticodon complex can be prevented by the selection
of synonymous codons.
In parallel with the selection of synonymous codons, the selection of anticodon wobble bases can
make a substantial contribution to the prevention
of slippage of the A-site anticodon, since translational speci®city of most of the wobble bases is restricted. Only unmodi®ed A and U in the
anticodon wobble position of the correct duplexes
can recognize all four bases, U, C A and G, in the
third position of codons (Barrell et al., 1980;
Heckman et al., 1980; Sibler et al., 1986; Boren et al.,
1993; Inagaki et al., 1995). However, A almost
never exists in the ®rst anticodon position (Sprinzl
et al., 1991). The anticodon wobble U (the wobble
base of greatest abundance) is frequently and variously modi®ed and its modi®cations always restrict translational speci®city. The wobble U is
modi®ed even in tRNAs that translate the unmixed
codon families (where four codons specify the
same amino acid) despite the fact that any of these
families is potentially translatable by a single anticodon with an unmodi®ed wobble U, while tRNAs
with modi®ed wobble U cannot decode all four codons. All known wobble bases except A and U can
recognize only 1 to 3 codon bases. For example, 2thiouracil (s2U), unmodi®ed cytosine (C) and 2lysylcytosine (k2C) decode only A, G and A, respectively; G decodes U and C; 5-methyluracil derivatives (xm5U) decode A and G; 5-hydroxyuracil
883
derivatives (xo5U) decode U, A and G; inosine (l)
decodes U, C and A (Lim, 1994, 1995; for reviews,
see Agris, 1991; BjoÈrk, 1992; Osawa et al., 1992).
It is clear that the selection of synonymous codons
and anticodon wobble bases is capable of counteracting slippage of the P-site anticodon. According
to the tandem slippage rule, the P-site tRNA, besides the correct duplexes, can form any allowed
duplex in which the pair formed by the ®rst anticodon base is not ®xed within the mini-helix. The allowed group of codon ± anticodon duplexes, as
noted above, is about 30% of all conceivable duplexes. This means that at uniform distribution of
codons in coding sequences the probability of the
correct duplex ! any allowed duplex transition resulting from slippage of the P-site tRNA should be
0 3 on the average. This probability can be reduced by the selection of synonymous codons and
anticodon wobble bases counteracting the formation of the allowed duplexes and mobile wobble
base-pairs. However, full elimination of the correct
duplex ! any allowed duplex transition at the Psite is impossible because the selection of synonymous codons and anticodon wobble bases can
control a wobble in the pairing of only two codon
bases, whereas in allowed duplexes a wobble in
the base-pairing of all three codon bases is possible
when the codon is not ®xed in the A-form by the
AP interduplex interaction.
Contrary to the correct duplex ! any allowed duplex transition at the P-site, the probability of the
correct duplex ! correct duplex transition at the
A-site can be reduced practically to zero by the selection of synonymous codons and anticodon wobble bases because a wobble in the base-pairing in
the correct duplexes occurs only at the third codon
position. Though it should be noted that in rare
cases a slippage of the A-site anticodon cannot be
prevented by the selection of synonymous codons
and anticodon wobble bases. For example, when
an amino acid sequence contains Met and Trp
(minor amino acid residues). In contrast to the
other amino acid residues, each of these residues is
speci®ed by a single codon (Met is speci®ed by
AUG and Trp by UGG). Therefore, for example,
ÿ2 and ÿ1 slippages of the A-site anticodon interacting with the codon GGG in the sequence 50 UGG-GGG-30 that codes Trp-Gly cannot be prohibited by the selection of synonymous codons and
anticodon wobble bases. But in such cases tandem
slippage can be prohibited by the elimination of
the correct duplex ! any allowed duplex transition at the P-site. Thus, we see that in the
presence of the AP interduplex interaction, frameshifting can be prevented within coding sequences
by the selection of synonymous codons and anticodon wobble bases.
It is apparent that within the framework of our
model the selection of synonymous codons should
occur to prevent frameshifting and it could be one
of the reasons why the contexts of both sense codons and stop codons are non-random (Ikemura,
1981; Kohli & Grosjean, 1981; Lipman & Wilbur,
884
1983; Yarus & Folley, 1985; Shpaer, 1986; Gouy,
1987; Hanai & Wada, 1989; Andersson & Kurland,
1990; Hat®eld & Gutman, 1992). The synonymous
codon selection allowing avoidance of tandem slippage may partially explain 3-1 and 3-3 doublet
bias (the doublets consisting of the third base of
one codon and the ®rst or third base, respectively,
of the following codon), which has been demonstrated in coding sequences (for reviews, see Yarus
& Curran, 1992; Buckingham, 1994).
The PE interduplex interaction
The AP interduplex interaction should be retained
during ribosomal translocation and in the posttranslocation state in the PE codon ±
anticodon complex
In the absence of the AP interduplex interaction,
the correct duplex ! any allowed duplex transition resulting from tRNA slippage can occur at
the P-site and at the A-site. As noted in the preceding section, the selection of synonymous codons
and anticodon wobble bases counteracts the correct
duplex ! any allowed duplex transition, but full
elimination of such transitions within coding sequences is impossible. This suggests that the AP interduplex interaction should be retained during
ribosomal translocation and in the post-translocation state, since in the absence of a counteractive
mechanism, frameshifting can occur in the PE
tRNA-mRNA-tRNA complex. Speci®cally, prokaryotic ribosomes recode the HIV-1 gag-pol-1 frameshift sequence by an E/P site post-translocation
simultaneous slippage mechanism (Hors®eld et al.,
1995). The assumption that the AP interduplex
interaction should be retained is strongly supported experimentally by the work of Nierhaus
et al. (1995), in which it is shown that the mutual
arrangement of the A and P-site tRNAs does not
change strongly during translocation. Retention of
the AP interduplex interaction means that the PE
interduplex interaction that is identical with that of
AP should exist. In thise case, the AP interduplex
interaction will take place throughout the ribosomal elongation cycle and so prevent slipping frameshifting caused by tandem slippage. Therefore,
we hypothesize that the PE interduplex interaction
is identical with that of AP. In this case, properties
1 to 5 of the AP interduplex interaction should be
true also for the PE interduplex interaction.
The AE interduplex indirect linkage
The simultaneous existence of the A and E-site
codon ± anticodon duplexes should be prohibited
by an indirect linkage between them
In the APE codon ±anticodon complex the relative
position between the A and E-site codon ±anticodon duplexes can be described as a right-handed
Interactions Between Codon ±Anticodon Duplexes
helical displacement of the A-site duplex to E site:
a rotation by 2 100 around the helix axis and a
Ê
simultaneous shift by approximately 2 8 A
(Figure 5). At such a relative position the A-site duplex is not in direct physical contact with the E-site
duplex. However, the AP and PE interduplex interactions provide an indirect linkage between these
duplexes. The PE interduplex interaction ®xes the
P-site codon (property 1) and, consequently, the Psite wobble pair. In turn, a ®xation of the P-site
wobble pair counteracts the formation of both the
correct and the wrong codon ± anticodon duplexes
at the A-site (property 4). Hence, the AP and PE interduplex interactions should destabilize the APE
codon ± anticodon complex.
The most likely way of eliminating the destabilizing in¯uence of the interduplex interactions is to
expel a codon ±anticodon duplex from the A or Esite. With regard to the P-site duplex, its disruption
is accompanied by the simultaneous breaking of
about six base-base and inter-ribose hydrogen
bonds as a result of holding of the codon ends by
the A and E-site duplexes. The simultaneous breakdown of six hydrogen bonds creates the barrier,
the overcoming time t ˆ eE/kT 10ÿ13 second of
which at E 6 20 kJ/mol and T ˆ 300 K lies between 107and 108 seconds. This time is much greater than that of the elongation cycle (10ÿ2 to 10ÿ1
second; Spirin, 1986). All this means that the simultaneous existence of the A and E-site codon± anticodon duplexes should be prohibited via the AP
and PE interduplex interactions (the AE interduplex indirect linkage).
The AE interduplex indirect linkage correlates well
with experiment. Direct visualization of three deacylated tRNAPhe molecules in the ribosome
(Agrawal et al., 1996) has revealed that the anticodons of the A and P-site tRNAs may interact with
two adjacent codons, whereas the position of the
E-site tRNA appears to exclude codon± anticodon
contact. This result supports the main consequence
of the AE interduplex indirect linkage, that the
simultaneous existence of the A and E-site duplexes should not take place, as well as a conclusion (Robertson et al., 1986) that the E-site is
kinetically heterogeneous, exhibiting more than
one successive state for the anticodon region of a
transiting tRNA molecule.
Because of the AE interduplex indirect linkage an
occupation of the A-site in the ribosomal pre-translocation state should reduce the af®nity for tRNA
of the E-site and vice versa in the post-translocation
state. Accordingly, the elongating ribosome should
always carry two tRNA: at A and P-sites before
translocation and at P and E-sites after translocation. This is entirely consistent with the allosteric
three-site model of elongation (Rheinberger &
Nierhaus, 1986; Gnirke et al., 1989; Triana-Alonso
et al., 1995; for reviews, see Rheinberger, 1991;
Nierhaus, 1993), and the AE interduplex indirect
linkage can be considered as a molecular mechanism explaining the allosteric interaction between
885
Interactions Between Codon ±Anticodon Duplexes
Limitations of the approach and its
further elaboration
Figure 5. A representation of the APE codon± anticodon
complex. The curved tube is mRNA (the A, P and E-site
codons). Acute angles are the anticodon loops of the A,
P and E-site bound tRNAs forming codon ± anticodon
duplexes with mRNA. Broken lines show the wobble
base-pairs. The P-site wobble pair interacts with the Asite codon ± anticodon duplex (the AP interduplex interaction), and the E-site wobble pair interacts with the Psite duplex (the PE interduplex interaction). The A, P
and E-site codons form a helix. The axis of this helix is
the long vertical arrow (the axis of the dihedral angle o,
see Figure 1(a)). The relative position between adjacent
codon ± anticodon duplexes can be described as a rotation by 100 around the helix axis and a simultaneous
Ê.
shift by approximately 8 A
The main limitation of the approach is that in the
proposed model ``outside'' factors affecting the formation and existence of the AP and PE codon ± anticodon complexes are not considered. There is a
broad spectrum of such outside factors: spatial
structures in a mRNA molecule; the af®nity of
tRNA molecule for the ribosomal sites including
the af®nity of a nascent peptide; aminoacyl-tRNA
imbalance; the selection of the wobble base-pairs;
non-standard structures of the anticodon loop; conserved purine base 37 in tRNAs, and so on. The action of these factors leads to non-slipping and
slipping frameshifting, ribosome hopping, misreading of the codon, abortive termination of translation (e.g. see Curran & Yarus, 1987; 1989; Jacks
et al., 1988; BjoÈrk et al., 1989; Andersson &
Kurland, 1990; Jacks, 1990; Chamorro et al., 1992;
Gesteland et al., 1992; Morikawa & Bishop, 1992;
Osawa et al., 1992; Peter et al., 1992; Tu et al., 1992;
Yarus & Curran, 1992; Hagervall et al., 1993;
Somogyi et al., 1993; Mottagui-Tabar et al., 1994;
Vimaladithan & Farabaugh, 1994; Atkins &
Gesteland, 1995; Barak et al., 1996; Chen et al.,
1996). In many cases the action of outside factors
can be explained within the framework of our
model. The action of certain of the factors on the
AP and PE codon ± anticodon complexes is analyzed in the following ( a comprehensive analysis
is beyond the scope of this work and will be
published elsewhere).
Outside factors leading to codon misreading and
the occurrence of fixed wobble base-pairs can
stimulate frameshifting via interduplex interactions
the A and E sites on the detailed molecular level in
terms of concrete atomic interactions.
The wobble pair of the E-site duplex can affect the
rate of codon reading at the A-site via the AE
interduplex indirect linkage
Stabilization or destabilization of the PE codon ±
anticodon complex will increase or decrease, respectively, the lifetime of the AE interduplex indirect linkage and so affect codon reading. However,
in these cases the in¯uence only on the rate of the
A-site occupation can take place. A pronounced effect on the accuracy of codon reading should be
absent because the AE interduplex indirect linkage
counteracts the formation of both the correct and
the wrong codon ± anticodon duplexes. If it is taken
into account that the stability of the PE codon ± anticodon complex depends on the mobility of the Esite wobble pairs, solvent molecules and modi®cations at their major edges (property 4), it is quite
obvious to think that the wobble pair of the E-ste
duplex can affect the rate of codon reading at the
A-site via the AE interduplex indirect linkage.
There are different outside factors responsible for
misreading of a codon. For example, aminoacyltRNA imbalance or an elevation of the aminoacyltRNA af®nity for the A-site caused by aminoacyltRNA mutations outside the anticodon (in this case
a lowering of the af®nity caused by the formation
of the wrong A-site duplex can be compensated).
The ®xed wobble base-pairs can be obtained, for
example, by the selection of synonymous codons
and the anticodon wobble base or by changing the
anticodon loop structure (see property 4). The appearance of the wrong codon± anticodon duplexes
at the A-site and the ®xed wobble base-pairs in the
P-site duplex should result in destabilization of the
AP codon ±anticodon complex in the pre-translocation state and the PE codon ± anticodon complex in
the posttranslocation state (see properties 2 and 4).
The destabilization of the AP or PE codon ± anticodon complex can be eliminated by tandem slippage or by expelling either of the two duplexes
formed by deacylated tRNA and peptidyl-tRNA
from the ribosomal A or P or E-site. In the ®rst
case, slippage of tRNA molecules should satisfy
the tandem slippage rule. In the second case, the
exit of the deacylated tRNA anticodon ± codon du-
886
Interactions Between Codon ±Anticodon Duplexes
plex from the P-site or E-site permits single slippage of peptidyl-tRNA. Because of the lack of the
interduplex interaction, the probability of single
slippage should be comparatively high (property
3). Thus, we see that misreading of the codon and
the small mobility of the wobble base-pair in the
P-site duplex can initiate slipping frameshifting
resulting from tandem slippage of deacylated
tRNA and peptidyl-tRNA or from single slippage
of peptidyl-tRNA.
tRNA conserved purine base 37 as an outside
factor affecting codon reading and frameshifting
In all elongator tRNAs position 37 next to the 30
end of the anticodon contains purine bases
(Grosjean et al., 1995). In the crystal structure of
tRNA (Jack et al., 1976) the second and third anticodon residues together with residue 37 are in the
A-form conformation. Owing to such an orientation, conserved purine base 37 simultaneously
stacks on the ®rst codon base and the third anticodon base (Figure 6) and so counteracts the large
propeller twist in the base-pair formed by these
bases. This, in turn, counteracts the formation of
non-canonical base-pairs in the codon ®rst position, since the large propeller twist is required in
many non-canonical base-pairs to avoid an uncompensated loss of hydrogen or polar atom ±ion
bonds (Lim, 1994, 1995). All this can serve as an
explanation for the fact that position 37 in all elongator tRNAs contains a purine base.
Apart from the fact that conserved purine base 37
counteracts the formation of non-canonical basepairs in the codon ®rst position, the type of purine
base 37 and its modi®cations determine the stability of codon ± anticodon duplexes and so affect
codon reading and frameshifting. Let us use unmodi®ed guanine at tRNA position 37 to illustrate evidence supporting this conclusion (a comprehensive
analysis of the conserved purine base 37 properties
will be published elsewhere).
At tRNA position 37 the guanine NH and NH2
groups together with solvent molecules form two
hydrogen bonds, which in the codon ±anticodon
duplex are stacked on the ®rst codon base when
residue 37 is in the A-form conformation (Figure 6).
Each of the solvent molecules forming these
stacked hydrogen bonds loses one of its 2± 3 hydrogen bonds, which can be formed with other solvent molecules in the absence of stacking. There
are three conceivable ways by which a loss of the
hydrogen bonds can be precluded: (1) shifts of
stacked hydrogen bonds toward the codon base
edge; (2) substitution of the ribosomal material for
the solvent molecules forming stacked hydrogen
bonds; (3) pairing of guanine 37 with a mRNA
base, i.e. the formation of the allowed mini-helix
extended by the base-pair G37 mRNA base (primarily by GC).
The ways (1) and (2) are unlikely. The way (1) is
not suited for guanine because two stacked hydrogen bonds of guanine in the vicinity of the A-form
Figure 6. Stacking pattern of bases in a double helix
demonstrating that a purine base in chain position i (in
this case guanine, bold lines) always simultaneously
stacks on both bases of the pair formed by the i ÿ 1th
base and its partner (the pair UA, ®ne lines positioned
under the bold lines). Three extrabold lines are glycosyl
bonds. Dotted circles are solvent molecules: water molecules and anions that can be considered as hydrogen
bond acceptors. These solvent molecules form hydrogen
bonds with the guanine NH and NH2 groups. The
hydrogen bonds are stacked on uracil. Guanine can be
considered as tRNA purine base 37 that is simulataneously stacked on the ®rst codon base and third anticodon base.
conformation cannot be forced out of stacking simultaneously. The way (2) is unlikely because tRNA
molecules must move in the ribosome. As for the
way (3), the formation of the extended mini-helix
at the A-site can occur through expelling the duplex from the P-site. In this case the exit of the duplex from the P-site allows avoidance of steric
restrictions within the P/A boundary of mRNA.
All this means that guanine at tRNA poition 37
can play the role of an outside factor, the action of
which leads to ‡1 non-slipping frameshifting. Consequently, unmodi®ed guanine at tRNA position
37 should be rare or absent and modi®cations in
its derivatives should eliminate one or both
stacked hydrogen bonds (a purine base forming
one stacked hydrogen bond, for example adenine,
is admissible because a single hydrogen bond can
be forced out of stacking).
These conclusions are entirely consistent with experiment. In contrast to unmodi®ed adenine, unmodi®ed guanine almost never exists at tRNA
position 37. There are only two known examples of
the presence of unmodi®ed guanine at position 37
and in all guanine derivatives one or both stacked
hydrogen bonds are eliminated by nucleoside
modi®cations (see the compilation summarising
the occurrence of modi®ed nucleosides by Grosjean
et al., 1995). As to frameshifting, there is direct evidence that methylation of the NH group of quanine (m1G) at tRNA position 37 prevents
frameshifting. In the Salmonella typhimurium trmD
mutant, which lacks m1G, the low-level ‡1 frameshifting caused by the de®ciency has been interpreted to be due to base-pairing between tRNA
base 37, G, and the ®rst codon base, C (BjoÈrk et al.,
1989; Hagervall et al., 1993).
887
Interactions Between Codon ±Anticodon Duplexes
The interduplex interactions and
programmed frameshifting
The chance of a shift in the reading frame is well
below 10ÿ4 (Atkins et al., 1972), but there exist
special sites in mRNA at which very high frameshifting frequencies have been detected (programmed frameshifting; for a review see, for
example, Atkins & Gesteland, 1995). Besides frameshift sites, programmed frameshifting is controlled
by special signals coded in mRNA. These signals
``stimulate'' high levels of shifting at the localized
shift site. There is a broad spectrum of stimulatory
signals that can be realized at frameshift sites
through the interduplex interactions. In fact, in the
above sections we have considered outside factors
playing the role of stimulatory signals (small mobility of the wobble pairs, misreading of the codon,
guanine at tRNA position 37) that can initiate frameshifting. In other words, within the framework
of the model of interacting codon± anticodon duplexes it is possible to attempt to design arti®cial
systems of programmed frameshifting in terms of
concrete atomic interactions and these systems can
be subjected to direct experimental tests.
Main results
Analysis of interactions between the codon ± anticodon duplexes gave the following main results.
Firstly, the AP interduplex interaction and not the
differences in the stability of the allowed duplexes
selects the correct codon ± anticodon duplexes: at
the A-site only the allowed duplexes with the Aform conformation of the codon are compatible
with the AP interduplex interaction and such duplexes should be considered correct.
Secondly, the AP interduplex interaction should be
retained during ribosomal translocation and in the
post-translocation state in the PE codon± anticodon
complex. The retention of the AP interduplex interaction throughout the ribosomal elongation cycle
and the selection of synonymous codons and anticodon wobble bases are able to prevent frameshifting within coding sequences. The synonymous
codon selection allowing avoidance of frameshifting may partially explain 3-1 and 3-3 doublet bias,
which is observed in coding sequences.
Thirdly, tandem slippage of peptidyl-tRNA and
deacylated tRNA can occur when peptidyl-tRNA
after slippage forms only correct duplexes, and
deacylated tRNA forms any allowed duplexes in
which the pair formed by the ®rst anticodon base
is not ®xed within the mini-helix (tandem slippage
rule).
Fourthly, there is a broad spectrum of factors leading to destabilization of the AP or PE codon± anticodon complex: small mobility of the wobble pairs,
misreading of the codon, unmodi®ed adenine at
the ®rst anticodon position, unmodi®ed guanine at
tRNA position 37, non-standard structures of the
anticodon loop, spatial structures in a mRNA molecule, and so on. The destabilization of the AP or
PE codon± anticodon complex can lead to frameshifting, since the destabilization of these complexes can be eliminated by tandem slippage or by
expelling either of the two duplexes formed by
deacylated tRNA and peptidyl-tRNA from the ribosomal A or P or E-site. In the ®rst case, tRNA
slippage should satisfy the tandem slippage rule.
In the second case, the exit of the deacylated tRNA
anticodon ± codon duplex from the P-site or E-site
permits single slippage of peptidyl-tRNA. Because
of the lack of the interduplex interaction, the probability of single slippage should be comparatively
high, as new codon ±anticodon duplexes formed
by peptidyl-tRNA need not be correct.
Fifthly, the indirect linkage between the codon ±anticodon duplexes bound to the A and E-sites follows from the AP and PE interduplex interactions.
This AE interduplex indirect linkage counteracts
the simultaneous existence of the A and E-site duplexes: the occupation of the E-site counteracts the
formation of any duplex at the A-site and, conversely, the occupation of the A-site is accompanied
by expelling the codon ± anticodon duplex from the
E-site.
Sixthly, the wobble pairs of the P and E-site duplexes can affect the rate of the A-site occupation
by aminoacyl-tRNA via the AP interduplex interaction and the AE interduplex indirect linkage, respectively.
Seventhly,anticodonwobbleresidue modi®cations
can take part in the prevention of frameshifting
and in the regulation of the rate of the A-site occupation by aminoacyl-tRNA. Probably, for this
reason the anticodon wobble residues are frequently and variously modi®ed. For example, the
anticodon wobble U (the wobble base of greatest
abundance) is modi®ed, as a rule, even in tRNAs
that translate the unmixed codon families (where
four codons specify the same amino acid), despite
the fact that any of these families is potentially
translatable by a single anticodon with an unmodi®ed wobble U, while tRNAs with modi®ed wobble
U cannot decode all four codons.
Finally, purine base 37 in tRNA molecules simultaneously stacks on the ®rst codon base and the
third anticodon base and so counteracts the large
propeller twist in the base-pair formed by these
bases. This, in turn, counteracts the formation of
non-canonical base-pairs in the codon ®rst position, since the large propeller twist is required in
many non-canonical base-pairs to avoid an uncompensated loss of hydrogen or polar atom ±ion
bonds. All this can serve as an explanation for the
fact that position 37 in all elongator tRNAs contains a purine base.
Materials and Methods
Stereochemical analysis was made using the FRODO
program on an Evans & Sutherland PS390 computer
graphics system and Corey-Pauling-Koltun atomic
models. The experimentally observed level of misreading
and frameshifting in vivo is 10ÿ4 to 10ÿ3 (Atkins et al.,
888
1972; Spirin, 1986; Parker, 1989; Jùrgensen & Kurland,
1990). This means that if the selection of tRNAs rests
only on the difference in the free energy of complex formation, at equilibrium a difference of G 20 kJ/mol
between wrong and correct association is required (the
probability of misreading or frameshifting exp(ÿG/kT)
at G 20 kJ/mol and T ˆ 300 K is 5 10ÿ4). Therefore,
in considering interactions between the codon ± anticodon duplexes and in the search for allowed structures, a
complex was disallowed when an uncompensated loss
of hydrogen or polar atom ± ion bonds was observed
and/or when at least one interatomic distance was less
than the extreme limit (rarely observed steric overlap).
Such a disallowed steric overlap and a polar atom ± ion
bond are comparable to the hydrogen bond in energy
(the energy of a normal hydrogen bond is about 20 kJ/
mol). The contribution of the other interactions (interactions in the sugar-phosphate backbone, base stacking
stabilized mainly by dipolar and London dispersion
forces and hydrophobic effects) was ignored, since these
interactions are an order of magnitude weaker than a hydrogen bond. For details and references, see Materials
and Methods (Lim, 1994).
Acknowledgements
The author is grateful to Drs J. F. Curran, M. B. Garber
and K. H. Nierhaus for constructive critical comments
on an earlier version of the manuscript and to Drs R.
Brimacombe and J. Frank for kindly providing the data
on the arrangement of tRNAs within the ribosome. This
work was supported by grant 96-04-48774 from the
Russian Foundation for Basic Research
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Edited by D. E. Draper
(Received 13 May 1996; received in revised form 31 October 1996; accepted 31 October 1996)