[48] C.C. Correll, B. Freebom, P.B. Morel, T.A. Steitz, Cell 91 (1997) 705.
[49] M. Brandl, M. Meyere, J. Suhnel, J. Phys. Chem. A 104 (2000) 11177.
CHAPTER SIX
MOLECULAR MECHANICS STUDIES ON
HYDROGEN-BONDED DUPLEXES
BETWEEN CODONS AND ANTICODONS
VI.1 Introduction
VI.1.1 Anticodon Stem-loops
VI.1.2 Helical and Stacking Interaction Parameters
VI.1.3 Backbone Torsional Angles
VI.1.4 Previous Experimental and Theoretical Studies
VI.1.5 Generalized Born Model for Solvation
VI.2 Computational Methodology
VI.2.1 Molecular Mechanics
VI.2.2 Amber 10 Force Fields
VI.2.3 Modeling Starting Structures
VI.2.4 Creating prmtop and inpcrd Files
211
VI.2.5 Control of Minimizations
VI.2.6 Structural Markers for Duplexes
VI.2.7 Pairing Energies of Duplexes
VI.3 Results and Discussion
VI.3.1 Duplexes with Guanine as AWB
VI.3.2 Duplexes with Uracil as AWB
VI.3.3 Duplexes with Cytosine as AWB
VI.4 Conclusions
References
VI.1 Introduction
Accurate gene expression is dependent on the high fidelity of the genetic code,
where the genetic information encoded in mRNA (as copied from DNA) is
translated in a highly controlled manner on to the amino acid sequence of the
protein synthesized. The translational decoding process involves recognition and
acceptance of hydrogen-bonded pairing between a specific trinucleotide
sequence of mRNA (the codon) and a corresponding trinucleotide sequence of
tRNA (the anticodon) often present in positions 34 to 36 of the seven nucleotidelong anticodon stem-loop. Such base pairings result in a short double-helix called
the codon-anticodon minihelix. Crystallographic studies [1,2] have revealed that
these codon-anticodon pairings take place in the proximity of the A site of the
30S ribosomal subunit (the decoding centre). Three 16S rRNA ribosomal bases of
the A site, viz., the nucleotide G530 (from the shoulder domain of the A site) and
the unpaired A1492 and A1493 (called universally conserved adenines of the
helix H44), interact with the codon-anticodon minor groove, dictating that the
212
first two base pairs of the codon-anticodon helix to be strictly of the WatsonCrick type (A:U, U:A, G:C and C:G).
It has recently been established that the A-minor motif (the most abundant
non-Watson-Crick tertiary interaction in the large ribosomal subunit) is
efficiently utilized by the small ribosomal subunit to discriminate between
cognate and near cognate tRNA. The bases A1492 and A1493 of the 16S rRNA
playing a major role in reading the shape of the first two base pairs of the codonanticodon helix [3]. However, it is believed that such contacts of the ribosome at
the wobble position of the codon-anticodon helix do not depend much upon the
precise shape of the codon-anticodon minor groove [1]. As a result, certain nonWatson-Crick base pairs (wobble base pairs) are permissible at this position. This
wobble base pairing allows a single tRNA to recognize several different codons,
an important feature linked to the degeneracy of the genetic code.
These recent findings are in fine agreement with Crick’s wobble hypothesis [4]
where, for the first time, it was pointed out that the degeneracy of the genetic
code arises due to a certain looseness or ambiguity during the hydrogen-bonded
base pairing between the third base of the codon and the first base of the
anticodon (the wobble bases), although the first two bases of the codon pair
with the last two bases of the anticodon in normal Watson-Crick double-helical
fashion.
Various factors may dictate the presence of a given base pair at the wobble
position. The first factor is similarity in configuration of the given wobble pair to
the Watson-Crick alignment (assumed as optimal for the mini-helical situation of
the codon-anticodon pair). Other conceivable factors may include the influence
of the other two (non-wobble) bases in the codon and anticodon, as well as the
influence of extra-codonic and extra-anticodonic base sequences. Pairing at the
wobble position is also heavily influenced by tRNA nucleoside modifications [5].
A detailed comparison of pattern and machinery involved in various steps of the
213
translation process in Eukaryotes, Bacteria and Archaea reveals that the fundamental events of protein biosynthesis are the same, but the ways they are
achieved in the various kingdoms of life often differ considerably [6].
Chapters Three, Four and Five of this Thesis have treated only the first factor,
and adhere to the hypothesis that pairing between a codon and anticodon
having the same non-wobble pairs depends primarily on the configuration of the
wobble pair alone. Nevertheless, one proceeds here beyond the solitary wobble
pairs (studied in Chapters Three, Four and Five) to investigate the entire
trinucleotide sequences of the codons and the anticodons. This approach is far
more realistic than the limited study at the level of the solitary wobble pair
alone. It remains to be seen whether the preliminary findings obtained at the
level of the solitary wobble pair are sustained and conserved at the level of the
complete trinucleotide H-bonded complexes between codons and anticodons.
One focuses here only on codon-anticodon complexes which involve
three major RNA bases functioning as anticodon wobble bases (AWB),
viz., guanine (Gua), cytosine (Cyt) and uracil (Ura). As state earlier,
adenine (Ade) never occurs as an AWB. For the time being, and for the
purpose of this Dissertation, no minor RNA bases functioning as AWBs
are taken for study here yet. Various anticodon triplets which have Gua,
Cyt or Ura at the AWB position are each paired here with a number of
codon triplets, subject to the condition that the non-wobble H-bonded
pairs of each duplex are all of the strict Watson-Crick type. This study
thus incorporates both cognate and near-cognate cases of codon-anticodon
pairing. The codon-anticodon duplexes investigated here include those
which are allowed in nature as well as some disallowed during translation.
214
Codons and anticodons always pair in an antiparallel fashion, where the
base sequence of the codon triplet is reckoned as proceeding from the 5'
end to the 3' end, while the base sequence of the anticodon triplet as its
pairs with the codon is read reverse to this, namely from the 3' to the 5'
end. Antiparallel pairing is also characteristic of all nucleic acid paired
segments, whether in DNA or RNA or during mRNA synthesis. In this
study, only the cognate and near-cognate cases are considered. Nearcognate cases involve pairs which are not allowed in nature, but
nevertheless have only Watson-Crick type base pairs at the two nonwobble positions. Only the wobble position contains non-canonical base
pairs, and the duplex may be allowed or disallowed depending upon
whether the wobble pair is allowed or disallowed. Non-cognate duplexes
are commonly regarded as those which have non-canonical base pairs at
the two non-wobble positions. Since only the four major RNA bases are
found at the codon wobble position, only four different codons are
available for H-bonded pairing with any one anticodon, since the bases at
the other two (non-wobble) positions are fixed for these cases.
Fig. VI.1 (next page) depicts the four pairing schemes involved in
codon-anticodon pairing with GCC as a sample anticodon. The wobble
position is marked by bold dark lines and the codon wobble base by
italicized letters. In order to arrive at cognate or near-cognate duplexes,
the anticodon GCC can in principle pair with the only four possible
codons GGC, GGU, GGA and GGG, giving rise to the four hydrogen
215
bonded trinucleotide duplexes GCC:GGC, GCC:GGU, GCC:GGA and
GCC:GGG. Of these four, the duplexes GCC:GGC and GCC:GGU are
observed in the context of protein synthesis, and involve the codons GGC
and GGU (with the G:C and G:U pairs allowed at the wobble position).
The duplexes GCC:GGA and GGC:GGG are not observed in nature during
translation, and involve the non-cognate codons GGA and GGG having
the disallowed G:A and G:G pairs at the wobble position.
5/
3/
5/
3/
5/
3/
5/
3/
G
C
G
U
G
A
G
G
C
G
C
G
C
G
C
G
C
G
C
G
C
G
C
G
5/
Anticodon
3/
Codon
5/
Anticodon
3/
Codon
5/
Anticodon
3/
Codon
5/
Anticodon
3/
Codon
Fig. VI.1 Four possible codon-anticodon pairs involving the anticodon GCC
Through this computational study, one attempts to investigate what the
precise pairing configurational factors are which allow only cognate
codons to pair with an anticodon during the translational decoding
process, while disallowed codons are excluded. Since the non-wobble
pairs in cognate and near-cognate triplet duplexes are of the canonical
Watson-Crick type, it must be the pairing configur-ation of the base pair
at the wobble position that discriminat es between allowed and disallowed
duplexes here. Although the role of the solitary wobble pair has been quite
successfully studied in Chapters Three, Four and Five, one deems that
investigation at the level of the complete trinucleotide duplexes would
216
provide a fuller picture. Using a well-parametrized classical molecular
mechanics model as the tool of study, one seeks to predict what really are
the energetic and configur-ational factors which discriminate between the
allowed and the disallowed codon-anticodon minihelices.
VI.1.1 Anticodon Stem-loops
Elements of tRNA structure which lie outside the actual triplet anticodon itself
may also be involved in fine tuning of the codon-anticodon recognition process,
although they may not in themselves contribute to the final outcome of inclusion
or exclusion of a given codon-anticodon pairing combination. Coding efficiency
of a particular anticodon is often influenced by the adjacent anticodon stem-loop
(ASL) nucleotides [7]. Various in vivo studies [8-10] and in vitro biochemical
studies [11-14] suggest that the conserved residues at positions 32, 37 and 38 in
the ASL affect translation efficiency and binding to the cognate codon. Residues
at positions 32 and 38 form a pseudo base pair at the top of the ASL[15], where
the nucleotide 37 (always a purine; G or A in Archaea and Eukaryae) is often
modified. The crystal structures of modified tRNA ASL's [16] have shown that in
order to bind the two lysine codons AAA and AAG to the anticodon UUU of
tRNA(Lys), it is essential that the modified nucleoside N6-threonylcarbamoyladenosine be present at position 37 in addition to either 5-methylaminomethyluridine or a 2-thiouridine residue at the wobble position. Thus, a bulky modified
nucleotide is used at position 37 to facilitate stacking interactions [17-19] as well
as to preposition the anticodon for interaction with the codon [19]. NMR analysis
of the individual effects of mnm5s2U34, s2U34, t6A37, and MgII on tRNA(Lys) ASLs of
E. coli [20] has shown how modifications at position 37 and 34 bring about a
non-canonical to a canonical structural transition.
217
VI.1.2 Helical and Stacking Interaction Parameters
The double-helix is the most common structural element of nucleic acids. Its
geometrical configuration is defined to a large extent by base-pairing and basestacking interactions along with the phosphodiester backbone conformation and
sugar puckers. All these interactions control the relative orientations and
positions of purine and pyrimidine bases along a given duplex strand. The mutual
orient-ation of the bases reflects not only their local sequential arrangements
but also the overall locus of the helical structure. For a more specific description
of the base-stacking and base-pairing interactions along the successive moieties
of a nucleic acid fragment, a set of helical parameters had been introduced by
some early workers [21-23]. More recently, methods like METHADON [24]
(MEthod for deTermination of HelicAl parameters using Dipolar cOupliNgs) have
received popularity, since they may be used to determine the helical parameters
directly from experimental data. The definition of these helical parameters is
treated in greater detail later on in this Chapter (see Section VI.2.6).
Base stacking is one of the driving forces responsible for the stabilization of the
three-dimensional structure of DNA and RNA. In contrast to the compositiondependent hydrogen-bonding energy of Watson-Crick base pairing, basestacking interactions are very sequence-dependent, as first confirmed from the
analysis of a dodecamer crystal structure [25]. A number of experimental and
theoretical methods have been utilized to investigate the base-stacking
phenomenon, and this is attributed to electrostatic interactions, hydrophobic
effects, and dispersion inter-actions [26-28]. As a result, a set of appropriate
base stacking parameters may be defined and utilized, as described later in
Section VI.2.6.
VI.1.3 Backbone Torsional Angles
218
The conformational versatility of RNA double helices depends much on the salt
concentration of their environment. The A-RNA double helix, a right-handed
conformer of RNA, predominates at lower salt concentrations, and exhibits
features typical of Watson-Crick DNA duplexes. It contains 11 base-pairs per turn
or 11 nucleotides within one helical pitch [29]. Specific RNA backbone conformations and interactions are crucial for RNA catalysis [30,31], for drug and
aptamer binding, and for protein/RNA interactions. As shown in Fig. VI.2 below,
there are six rotatable torsion angles per residue along the RNA backbone [32].
Due to the complex variability of these numerous torsion angles per residue,
techniques like X-ray diffraction and NMR have difficulty in determining the
backbone conform-ations except for very high resolution X-ray crystal structures,
seldom attainable for RNA molecules of biologically interesting size.
Fig. VI.2 Backbone torsion angles in nucleic acid structure
Table VI.1 below presents the average values of the various torsional angles (in
o
) as typically found for various types of nucleic acid helices [33].
219
Table VI.1 Average torsion angle values for various nucleic acid helices
__________________________________________________________________
α
Nucleic acid type
β
γ
δ
ε
δ
__________________________________________________________________
A-RNA
-68
178
54
82
-153
-71
A-DNA (fibres)
-50
172
41
79
-146
-78
B-DNA (fibres)
-41
136
38
139
-133
-157
Z-DNA (C residues)
-137
-139
56
138
-95
80
Z-DNA (G residues)
47
179
-169
99
-104
-69
__________________________________________________________________
*Taken from Ref. [33]
VI.1.4 Some Experimental and Theoretical Studies
Analysis of 100 complete sets of the cytoplasmic elongator tRN A genes
from Bacteria, Archaea, and Eukarya [34] suggested that the number of
the hydrogen bonds formed between the complementary nucleotides in the
anticodon–codon duplex appears as a major quantitative parameter
determining co-variations amongst Bacteria, Archaea, and Eukarya. Direct
observation of the wobble pairs (involving the anticodon wobble
nucleoside inosine) at the decoding center of a ribosome has now been
achieved by an X-ray crystal structure study [35]. Canonical and
mismatched RNA base pairs may both be easily adopted into RNA minihelices due to the mobility of the polynucleotide chain around the A-form
conformation. A wrong codon-anticodon duplex may thus yet be stable
220
enough to be observed under in vitro experimental conditions, as indeed
has been found to occur in a crystalline yeast tRNA mini-helix [36].
Progresses in the theory of intermolecular forces, solid state physics methods
and computer simulation techniques have helped us to understand various
physic-chemical processes in condensed phases and biological environments.
Recent crystal structures of the small ribosomal subunit have made it possible to
examine the detailed energetics of codon recognition on the ribosome by
computational methods. Notable work in this field includes a study of the
energetics of codon-anticodon recognition on the small ribosomal subunit [37], a
geometric analysis of mechanisms of discriminating between correct and
incorrect tRNAs by ribosome using molecular dynamics (MD) calculations in
explicit solvent [38], and study of the effect of codon-anticodon interaction on
the structure and dynamics of transfer RNAs using molecular dynamics
simulations over a nanosecond time scale [39]. The effect of the dielectric
constant of five different solvents in the displacement of amino acid sequences
on codon–anticodon residues in proteins have been studied using BLYP and
B3LYP/3-21G, 6-31G, and 6-31G* levels of theory [40].
The above survey reveals that much as yet needs to be carried out with respect
to the study of complete codon-anticodon pairs in solvent phase in the context
of explaining the specificity and degeneracy of the genetic code. Such studies, if
successful, may be expected to differentiate clearly between cognate and noncognate duplexes, including the disallowed near-cognate systems. This Chapter
presents some preliminary efforts along this line, hopefully to be augmented in
the near future here.
VI.1.5 Generalized Born Model for Solvation
221
Various properties of biological macromolecules, including geometry, vibrational
frequencies, total energy, electronic spectrum, relatively weak forces like van der
Waals, dispersion, hydrogen bonding interactions etc., all heavily depend on the
solvent around them. The presence of a polar solvent can even stabilize charge
separation within a molecule which in turn causes electron density shifts and
influences the associated properties of the molecule to a large extent. It has
been shown by Monajjemi et al. [40] that interactions between water molecules
and triplets like AAA, UUU, UUC and AAG reduce the energy of the whole
system.
Explicit solvation models require thousands of discrete water molecules to be
placed around the solute under study, which becomes computationally very
expensive. Upon replacing the discrete water molecules by “virtual water” (an
infinite continuum medium with some of the dielectric and “hydrophobic”
properties of water), the computational cost may be reduced. Advantages of the
implicit solvent continuum models over the explicit representations include short
time equilibration of solvent molecules, improved sampling, higher degree of
algorithm flexibility, and no artifacts of periodic boundary conditions. Estimation
of free energies becomes feasible since solvent degrees of freedom are taken
into account only implicitly. Continuum implicit solvent models have been found
to be successful in calculating various macromolecular properties in solution [4143].
Most molecular modeling applications involve computation of total energy of a
molecule in the presence of solvent, which is a function of molecular configuration. The total energy of a solvated molecule can be written as
Etot = Evac + ΔGsolv
where, Evac is the energy in gas phase and ΔGsolv is the solvation free energy.
222
To estimate ΔGsolv it is decomposed into the electrostatic and non-electrostatic
component parts:
ΔGsolv = ΔGel + ΔGnonel
where, ΔGnonel is the free energy of solvating a molecule without charges and
ΔGel is the free energy without charges in the vacuum, but added back again in
the presence of a continuum solvent environment. This decomposition strategy
is the standing ground for the widely used PB/SA scheme [44].
The analytic generalized Born (GB) method is an approximate way to calculate
ΔGel. This methodology has become popular due to its relative simplicity and
computational efficiency, compared to the more standard numerical solution of
the Poisson–Boltzmann equation [45,46].
VI.2 Computational Methodology
Calculation of the structure and energy of H-bonded trinucleotide duplexes is
clearly beyond the present scope of usual ab initio quantum chemical methods.
Fortunately, one may lay recourse to the various classical potential parametrized
force field methods which give good descriptions of the structure and energies of
macromolecules like nucleic acids, proteins and carbohydrate polymers. The work
of this Chapter makes use of such methodologies, commonly called molecular
mechanics, to treat the trinucleotide codon-anticodon duplexes studied here.
VI.2.1 Molecular Mechanics
In molecular mechanics the energy of a system is given by a force field
consisting of various bonded and non-bonded interaction terms. The potential
energy in the AMBER force field [47], is usually described as a summation of
bonded terms (bond stretching, angle bending, torsion angle) and non-bonded
terms (electro-static and van der Waals interactions). Thus the general expression
for the total energy determined by a given force field is as follows:
223
Etotal = Estretch + Ebend + Etorsion + Enon-bonded
The force field is used as an optimization criterion and the (local) minimum
searched for by an appropriate algorithm (e.g. steepest descent, conjugate gradient
etc). Force field methods have found wide-ranging popularity in structural studies
on biomacromolecules like nucleic acids, proteins, polysaccharides etc.
VI.2.2 Amber10 Force Fields
In molecular mechanics, a force field refers to the functional form and parameter
sets used to describe the potential energy of a system of particles (typically, but
not necessarily, atoms). Force field functions and parameter sets are derived from
both experimental work and high-level quantum mechanical calculations. "Allatom" (AA) force fields provide parameters for every atom in a system, including
hydrogen, while "united-atom" (UA) force fields treat the hydrogen and carbon
atoms in methyl and methylene groups as a single interaction center. By plotting
the RMS deviations of the minimized structures of about 25 different proteins as a
function of their crystallographic R factors of the initial structures, it has been
proved [48] that the use of all-atom models for energy minimization of proteins in
the AMBER force field is more accurate than the united-atom models. For the
systems studied here, one uses the FF99SB all-atom force field [49] containing
updated torsion terms for Phi-Psi angles (FF99SB) which improve the overestimation of alpha helices that occurs when using the FF94, FF96 and FF99 force
fields. However, the charges are still based on HF gas phase ab initio quantum
calculations and the bond angle and dihedral parameters are the same as the FF99
force field hence FF99SB and FF99 can be considered equivalent in this context.
VI.2.3 Modeling Starting Structures
The starting structures of the all the codon-anticodon pairs
involving major RNA bases are prepared using the nucgen module
built in the AmberTools 1.2 package. The nucgen program is
capable of generating the canonical A- and B- duplex geometries of
nucleic acids. In systems like the 3'GCC5'-3'GGG5' pair, where the
AWB guanine uses its Hoogsteen edge for wobbling, the nucgen
224
created structure is modified at the anticodon wobble position using
the GaussView and Open-Babel software packages. Negative
charges on the trinucleotide backbone (due to the phosphate
groups) of the nucgen created PDB structures are neutralized by
adding four explicit neutralizing Na+ ions for each anticodon-codon
pair after loading them in the xleap programme. There are a
number of different ways to add ions to a structure. One uses the
“addions” command implemented in xleap. This method works by
constructing a coulombic potential on a 1.0 angstrom grid and then
placing the counterions one at a time at the points of lowest or
highest electrostatic potential.
VI.2.4 Creating prmtop and inpcrd Files
The xleap routine is then used to create the prmtop and inpcrd files. The prmtop
file is the parameter/topology file, which defines the connectivity and
parameters. This information is static, or in other words, does not change during
the simulation. The inpcrd file is the coordinates (and optionally box coordinates
and velocities). This is data is not static and changes during the simulations
(although the file is unaltered).
VI.2.5 Control of Minimization
Since the steepest descent algorithm is suitable for quickly removing the largest
strains in the system but converges slowly when close to a minima, one used the
conjugate gradient method (controlled by the flag “maxcyc=50000”). The XMIN
method is used by specifying with the flag “ntmin=3” which by default uses a
Truncated Newton linear Conjugate Gradient method with optional LBFGS preconditioning. [50]. The periodic boundary condition is turned off by using the flag
“ntb=0”. Solvent effects are introduced using the “igb=1” flag which corresponds
to the "standard" pair-wise generalized Born model [51] using the default radii
set up by leap. This method has become popular [52-59] due to its relative
simplicity and computational efficiency, compared to the more standard
225
numerical solution of the Poisson-Boltzmann equation. Non-bonded cutoffs are
specified by the flag “cut= 999” meaning the calculation is carried out without a
cutoff.
VI.2.6 Structural Markers for Duplexes
The structures of codon-anticodon duplexes are estimated using various
geometry determinants viz., (a) Base Pair Configuration Markers, (b) Helical and
Stacking Interaction Parameters, and (c) Backbone Torsional Angles. The
following para-graphs briefly discuss these parameters:
(a) Base pairing configuration for each of the three base pairs of a codon-anticodon minihelix is separately estimated using the base pair configuration
markers as shown in Fig. IV.3 for the Gua:Cyt base pair as an example.
O
N
N
N
H
H
NH
N
C
N
N
HN
H
O
C
Rcc
N2
N1
C2
C1
1 = N1C1 C2
2 =  N2C2C1
c = C1N1N2C2
226
Fig. IV.3 Configuration markers for the Gua:Cyt base pair.
The distance Rcc between the carbon termini of the two would-be glycoside N-C
bonds of the two bases in the pair is related to overall width of the base pair, an
important factor often determining whether or not a given RNA base pair can be
accommodated during codon-anticodon pairing. The angles 1 and 2 between
these N-C bonds and the Rcc vector determine whether a given base pair would
be reversible or not, since similar 1 and 2 values mean that a pair X:Y would
have the same overall configuration as the reversed Y:X pair, as is true for the
DNA base pairs. The dihedral angle c spanning these two N-C bonds connected
by the Rcc vector indicates the degree of co-planarity of the two bases within the
pair, where greater co-planarity stabilizes the codon-anticodon pair as a whole
through promoting stacking interactions between adjacent base pairs.
OH
HO
N
N1
HN
ANTICODON
H
N
O
HO
O
H
H
C2
H
O
C5
NH
O
H
O
HO
H
H C3
H
N
H
N
N
H
NH
N
N3
N
O
O
HO
Helical Parameters
227
H
N
H
OH
N4
H
C4
H
O
P
OH
H
O
H
O-
O
H
O
O
5/
H
N5
O
H
P
O
H
O
N
N
H
HN
O
OH
H
N
N2
5/
-
C6
O
P
N
N
O
H
O
H
N6
O
HN
O
H
O
H
P
H
O
H
O
N
HO
O
N
C1
H
-
H
N
3/
H
NH
OH
CODON
N
H
H
O
N
O-
3/
d1= C2-C1-C6
e1= C1-C2
f1= [C2-C1-C6-C5]
d2= C1-C6-C5
e2= C6-C5
f2= [C3-C2-C5-C4]
d3= C3-C2-C5
e3= C2-C3
d4= C2-C5-C4
e4= C5-C4
Stacking Interaction Parameters
p1=N2-N1-N6
q1= N1-N2
r1= [N2-N1-N6-N5]
p2=N1-N6-N5
q2= N6-N5
r2= [N3-N2-N5-N4]
p3= N3-N2-N5
q3= N2-N3
p4= N2-N5-N4
q4= N5-N4
Fig. IV.4
Schematic representation of helical and stacking interaction
parameters of the GCC-GGC trinucleotide duplex as an example
(b) The geometrical configuration of a codon-anticodon mini-helix is estimated
using various helical and stacking interaction parameters as shown in Fig. IV.4
(previous page) for the GCC:GGC duplex as an example. These parameters are
represented by the various bond distances (viz., e1 to e4 for helical parameters
and q1 to q4 for stacking interaction parameters), angles (viz., d1 to d4 for
helical parameters and p1 to p4 for stacking interaction parameters) and
dihedral angles (viz., f1 and f2 for helical parameters and r1 and r2 for stacking
interaction para-meters) which are measured as per the schemes mentioned in
Fig. IV.4.
(c) The backbone torsional angles per residue along the RNA backbone as
earlier described in Fig. VI.2 have also been estimated for systems studied here
VI.2.7 Pairing Energies of Duplexes
228
The H-bonded pairing energies of a given trinucleotide codon-anticodon duplex
are expressed here as enthalpy terms, where the net pairing energy Ep for the
full system is given as
Ep = Et(duplex) – [Et(codon) + Et(anticodon)]
where the Et terms refer to the calculated total energies or enthalpies of
formation of the codon-anticodon H-bonded duplex, of the isolated codon and
of the isolated anticodon respectively as determined by the molecular mechanics
method. The standard packages for such methods are able to estimate energy
differences in terms of enthalpies or free energies. The pairing energies may, in
principle, be based upon the systems in gas (vacuum) phase or solvated
(aqueous) phase.
VI.3 Results and Discussion
During cognate codon-anticodon duplex formation the codon needs to maintain
the undeformed A-form of RNA [60]. However, stacking interactions between
bases or base pairs do not contribute to the codon anticodon recognition
process. Base stacking has been shown to be quite insufficient for promoting
duplex stability [61,62]. The stability differences introduced by various base
stacking motifs do not adequately differentiate between correct and wrong
codon-anti-codon duplexes. Such differences could, of course, be a factor which
distinguishes between various cognate and non-cognate duplexes. Table VI.2a
(next page) provides a list of the anticodon-codon pairs containing only the
major RNA bases and the amino acids they code for [63].
Table VI.2a List of anticodon codon pairs containing only the major RNA bases
and the amino acids they code for.
229
__________________________________________________________________
AWB Amino-acid Anticodon
Codons
Obsd pairs
Unobsd pairs
__________________________________________________________________
Gua
Cys
Gly
Cyt
GCC
UGU; UGC
GAU
UUU; UUC
Leu
GAG
CUU; CUC
GAA
G:U, G:C G:A, G:G
GGU; GGC
Ile
Phe
Ura
GCA
UUU; UUC
Ser
GCU
AGU; AGC
Thr
GGU
ACU; ACC
Val
GAC
GUU; GUC
Gly
UCC
GGA
Leu
UAG
CUA
Gln
CUG
CAG
Gly
CCC
U:A
Other pairs
C:G
Other pairs
GGG
Leu
CAA, CAG
UUG, CUG
Lys
CUU
AAG
Met
CAU
AUG
Trp
CCA
UGG
__________________________________________________________________
230
Table VI.2b (next page) furnishes a list of 12 anticodon-codon pairs modeled by
nucgen for this study. These 12 pairs arise out of the possible combinations of
three known anticodons (GCC, UCC and CCC) for the amino acid glycine as they
pair with a variety of codons (subject to the condition that the non-wobble base
pairs are of the true Watson-Crick type). The status as observed or unobserved
has been assigned to the duplexes depending on whether the duplexes formed
correspond to cognate or non-cognate (near-cognate) situations respectively.
This is related to the observed/unobserved status of the base pairs present at
the respective wobble positions as studied in the previous three Chapters.
Table VI.2b Anticodon-codon pairs based on the glycine anticodons with Gua,
Cyt and Ura as AWB giving cognate and near-cognate duplexes
__________________________________________________________________
No Anticodon
Codon
Duplex Type
WBP
Status
__________________________________________________________________
Guanine as AWB
1
GCC
GGA
NON
G:A (WC/WC)
Unobsd
2
GCC
GGG
NON
G:G (H/WC)
Unobsd
3
GCC
GGU
AW
G:U (WC/WC)
Obsd
4
GCC
GGC
WC
G:C (WC/WC)
Obsd
Cytosine as AWB
231
5
CCC
GGA
NON
CA (WC/WC)
Unobsd
6
CCC
GGG
WC
CG (WC/WC)
Obsd
7
CCC
GGU
NON
CU (WC/WC)
Unobsd
8
CCC
GGC
NON
CC (WC/WC)
Unobsd
Uracil as AWB
9
UCC
GGA
WC
U:A(WC/WC)
Obsd
10
UCC
GGG
NON
U:G(WC/WC)
Unobsd
11
UCC
GGU
NON
U:U(WC/WC)
Unobsd
12
UCC
GGC
NON
U:C(WC/WC)
Unobsd
__________________________________________________________________
These 12 anticodon-codon pairs can be divided into three classes – duplexes
containing standard Watson-Crick base pairs at the Anticodon Wobble Position
or AWP (denoted as WC), duplexes containing allowed non-canonical base pairs
at the AWP (denoted as AW) and duplexes containing disallowed base pairs at
the AWP (given as NON). Only the three duplexes GCC-GGC, CCC-GGG and UCCGGA can be considered as true WC type codon-anticodon mini-helices that exist
in all domains of life since they follow the canonical A-duplex geometries of
ribonucleic acids and contain only the standard Watson-Crick base pairs Gua:Cyt
and Ade:Ura (or their reverse). Only one duplex – the GCC:GGU – is of the AW
type, being allowed, but containing a non-Watson-Crick wobble pair. The 8 other
duplexes are all of the NON type, and are not allowed although they are all of
the near-cognate type. In this study, the configurations of all duplexes are
compared with the canonical WC type systems as a reference to gauge and
rationalize their observed or unobserved status.
232
Tables VI.3a, VI.4a and VI.5a (see later) present the interacting edges, the
pairing energies Ep and the values of the three base pair configurational markers.
Pairing energies of all the solvated duplexes are negative and range from -36.44
to -24.90 kcal mol-1. Relatively small values of pairing energies in solvent phase
for solitary RNA base pairs have been noted in previous DFT work by Sharma et
al. [64] and attributed to dampening of electrostatic interactions through
solvation. Since all duplexes have negative pairing energies, thermodynamic
facility of pairing is itself not sufficient to differentiate between observed and
unobserved duplexes, though it is necessary to ensure a stable pair. Canonical
and mismatched RNA base pairs may both be easily adopted into RNA minihelices due to mobility of the polynucleotide chain around the A-form
conformation. A wrong codon-anticodon duplex may thus be stable enough to
be observed experiment-ally, as found within a crystalline yeast tRNA minihelix
[36], although it would not be possible for it to occur in codon-anticodon pairing
during translation.
This suggests that the configuration of the duplex (especially around the
wobble base pair) is the real criterion for deciding whether or not the given
codon-anti-codon duplex is allowed or not during the translation process.
Suitability of the pairing configuration for each of the three constituent base
pairs may be gauged from values of the base pair configuration markers as
defined earlier above. The overall configuration and the geometry of the duplex
as a whole may be gauged from the helical and stacking parameters which have
been described earlier, and also from the backbone torsional angles.
The calculated Ep values for the three WC type duplexes are -36.44, -35.83 and
-31.26 kcal mol-1, considerably lower (larger negative values) than the other two
classes of duplexes, viz., the near-cognate AW or NON types. Thus these results
of the xmin optimization clearly indicates the stability order among these three
classes of duplexes is WC > AW > NON. The Rcc values for individual base pairs in
233
the WC type duplexes (10.67 to 10.75 Å), the 1 and 2 values (53.74 to 57.87)
and the dihedral c values (0.09 to 14.55) are all very close to the standard
values obtained for the free (solitary) canonical Watson-Crick pairs.
VI.3.1 Duplexes with Guanine as AWB
The 4 cognate and near-cognate trinucleotide duplexes arising out of the glycine
anticodon GCC having guanine as the AWB are depicted in Figs. IV.5a to IV.5d
below and in the succeeding pages. The codons GGA, GGG, GGU and GGC are
made to pair in antiparallel fashion with the anticodon GCC. The GCC-GGA and
GCC-GGG pairs are disallowed (being of the NON type), while the GCC-GGU pair
(of the AW type) and the GCC-GGC pair (WC type) are both allowed.
OH
N
N1
HO H
3/
ANTICOD
ON
O
HO H
C2
H
O
H
N
N2
H
O
P
O
O
HO H
H C3
H
N
O
N
N3
O
H
H
N
N4
N
NH
HO
Fig. IV.5a The GCC-GGA duplex
234
H
H OH
O
H
H
OH
H
C4
O
P
N
N
O
O
O
H
C5
O
O
H
H
O
N
N5
NH
H
5/
-
N
N
H
O
P
H OH
O
H
O
O
H
HN
O
C6
N
5/
-
O
H
NH2
N
P
H
O
H
O
H
O
N
N6
N
HN
O
N
N
N
HO
-
NH
N H
H C1
H
H
H OH
CODON
O
N
O-
3/
H2 N
NH
ANTICO
DON
H
H
H
N
HO H
P
C2
H
O
H
N
N2
P
O
O
O
H OH
O
HO H
H C3
H
N
N3
O
O
N
H
N
O
H
NH
H
N
N4
N
H OH
O
H
H
OH
H
C4
Fig. IV.5b The GCC-GGG duplex
O
P
N
HO
235
O
O
H
C5
O
O
H
H
O
N
N5
NH
H
5/
-
N
N
H
O
P
O
H
O
O
H
HN
O
H
H
N
5/
-
N
O
H
O
O
N
H
N
N6
C6
HN
O
H
O
N
H C1
HO
-
N
N
H OH
CODON
3/
H
N
N1
HO H
OH
O
O
N
O-
3/
O
HO H
3/
H C1
N
N1
N
ON
ANTICOD
HO H
P
O
C2
H
O
H
N
N2
H
O
P
O
O
H
HO H
H C3
H
N
O
N
N3
O
H
H
N
N4
N
NH
HO
Fig. IV.5c The GCC-GGU duplex
236
H
H OH
O
H
H
OH
H
C4
O
P
N
N
O
O
O
H
C5
O
O
H
O
N
N5
NH
H
5/
-
N
N
H
O
P
H OH
O
H
O
O
H
HN
O
O
H
N
5/
-
O
H
NH2
N
O
O
H
O
C6
HN
-
H
N
O
H
N
N
N6
HO
H
H
H OH
CODON
O
N
OH
O-
3/
OH
3/
H C1
N
HN
ANTICODO
N
HO H
P
O
H
N
O
O
C2
H
O
H
N
N2
P
O
C6
O
O
H
HO H
N
O
H C3
H
N
N3
O
H
H
H
N
N4
N
O
P
H OH
O
N
N
O
O
O
H
C5
O
O
H
O
N
N5
NH
H
5/
-
N
N
H
O
P
H OH
O
H
O
O
H
HN
O
O
H
N
5/
-
H
O
H
HN
-
N
N6
O
H
H
O
N
H
N
HO
H
NH
H
H
CODON
N
N1
HO H
H
O
N
O-
3/
OH
H
C4
NH
H OH
HO
Fig. IV.5d The GCC-GGC duplex
Table VI.3a (next page) presents the Ep values and the configurational data for
the four anticodon-codon pairs arising from the GCC anticodon (cognate for the
amino acid glycine) where the AWPs are all occupied by unmodified guanine
residues as depicted in Figs. IV.5a to IV.5d (previous pages). The GCC-GGA and
GCC-GGG duplexes are not observed, while the GCC-GGC and GCC-GGU duplexes
are observed. The first observation is that the allowed duplexes GCC-GGC and
GCC-GGU are associated with larger and more negative values of the pairing
energy Ep (-35.83 and -32.19 kcal mol-1 respectively) as compared to the two
disallowed duplexes GCC-GGA and GCC-GGG (for which the Ep values are -24.90
and -25.84 kcal mol-1 respectively). This shows that the observed duplexes are
definitely more stable than the unobserved duplexes. Even though
thermodynamic stability of the duplex per se is not a factor determining whether
237
or not the given duplex is observed or not, the trend definitely points to greater
stability of the allowed duplexes as compared with the disallowed ones.
Table VI.3a Configurational dataa of constituent base pairs within the anticodoncodon duplexes arising from the GCC anticodon having Guanine as the AWB after
energy minimization by xmin (solvated and neutralised with Na+ counter ions).
__________________________________________________________________
ACPs
BPs
Edges
Ep
Rcc
θ1
θ2
c
__________________________________________________________________
GCC-GGA
G:A (I)
wc/wc
(Unobsd)
C:G (II)
-24.90
12.86
47.61
49.28
1.39
wc/wc
10.81
55.34
53.90
8.50
C:G (III) wc/wc
10.61
58.95
54.01
15.64
11.57
30.01
67.75
2.18
GCC-GGG
G:G (I)
H/wc
-25.84
(Unobsd)
C:G (II)
wc/wc
10.78
55.61
53.37
9.58
C:G (III) wc/wc
10.68
57.85
54.03
12.20
10.15
45.44
75.44
0.33
GCC-GGU
G:U (I)
wc/wc
-32.19
(Obsd)
C:G (II)
wc/wc
10.77
55.70
54.58
5.89
C:G (III) wc/wc
10.43
61.20
55.12
7.19
10.72
54.29
56.63
12.39
GCC-GGC
G:C (I)
wc/wc
-35.83
(Obsd)
C:G (II)
wc/wc
10.75
56.10
53.87
14.55
C:G (III) wc/wc
10.69
57.62
54.02
12.64
__________________________________________________________________
a
Pairing energies in kcal/mol; distances in angstrom; angles in degrees
Furthermore, the configurations of the wobble pairs also strongly present
them-selves as discriminating factors which distinguish clearly between allowed
and disallowed duplexes. The wobble pair configurations for each case are given
in bold in Table VI.3a above. For the disallowed duplexes GCC-GGA and GCCGGG, the wobble pairs G:A and G:G are associated with longer values of the Rcc
marker (12.86 and 11.57 Å respectively). The allowed GCC-GGC and GCC-GGU
238
duplexes are, however, associated with values of the Rcc marker that are smaller
(10.72 and 10.15 Å respectively) and more in line with the standard WatsonCrick type of base pairing configuration.
239
GCC-GGA
GCC-GGC
240
GCC-GGG
GCC-GGU
Fig. VI.6 Three-dimensional representations of the optimized duplexes arising from GCC anticodon
241
Three-dimensional portrayals of the four optimized codon-anticodon duplexes
arising from the glycine GCC anticodon as obtained by AMBER are presented in
Fig. VI.6 (previous page) where the distinguishing characteristics of each duplex
may be visually recognized. These portrayals show the atoms as points and the
covalent bonds as differently coloured sticks. Hydrogen bonds are also depicted.
The base pairs at the wobble positions of the two NON type duplexes (GCCGGA and GCC-GGG) are G:A (wc/wc) and G:G (H/wc) respectively, while the
other base pairs at the two non-wobble positions are Watson-Crick base pairs.
The Rcc value of the non-canonical G:G (H/wc) base pair is 11.57 Å, the 1 and 2
values are 30.01 and 67.75 and dihedral c is 2.18. Judging from the context of
base pair width as determined in the work of the previous Chapters, the Rcc value
of this pair may be too large to allow it to occur at the wobble position. One
proposes that it may also be the large difference between the 1 and 2 values
that prevents G:G (H/wc) from occurring at the wobble position.
The disallowed G:A (wc/wc) pair in the unobserved NON type GCC-GGA duplex
also has an appreciably large Rcc value of 12.86 Å, although the values of the
other configuration markers are close to those of Watson-Crick base pairs, viz.,
the 1 and 2 values are 47.61 and 49.28 respectively and the dihedral c is
1.39. The large Rcc value appears to be the major discriminating factor here,
preventing the G:A (wc/wc) pair from occurring at the wobble position.
This study thus predicts that the configurations of the two NON type duplexes
(GCC-GGA and GCC-GGG) at the wobble position considerably deviate from
those of the WC duplex GCC-GGC and the AW duplex GCC-GGU. Although the
AW duplex GCC-GGU is less stable compared to the WC duplex GCC-GGC, the
configuration markers of the three bases have similar values for both duplexes,
except that the wobble base G:U has differing 1 and 2 values (45.44 and
75.44, differing by 30.0˚), showing that this base pair is not reversible between
strands. It has been well established that while a G 34-containing tRNA (guanine at
242
the AWP) can easily read U-ending codons by wobbling, the U34-containing
tRNAs read G-ending codons less efficiently. The efficiency of U34:G3 wobbling (U
at AWP and G at CWP) strongly depends on the presence of chemical adducts on
the C5 atom of U34 mediated by specific enzymes [65-67]. The lower stability of
the G:U pair compared to the A:U pair has also been noted [68,69], where
replacement of the A:U pair by G:U destabilizes the formation of doublestranded RNA by about tenfold.
The pyrimidine-ending codons (GGU and GGC) that code for the amino acid Gly
are always read by a G34-containing tRNAGly in Bacteria, Archaea and Eukarya
[70]. According to the revised wobble rules [5], an unmodified guanine residue at
the AWP can pair with a cytosine at the CWP to form the standard Watson-Crick
base pair G:C, while with a uracil at CWP, it forms the allowed wobble base pair
G:U [71]. Such a residue never forms the G:G or G:A pairs at the wobble position.
It is reported that a wobble G34:A3 base pair occurs in Euplotes (a ciliate
belonging to the phylum Ciliophora) which is used to read the rare sense codon
UGA3 (for the amino acid Cys) during mRNA translation [72]. However, such an
appearance of the G34:A3 base pair at the wobble position is very rare and has
not been reported in other domains of life.
Many extra-anticodonic factors like ribosomal monitoring, enzyme activities,
structure of the ASLs, genome size etc. may also contribute to prevent
occurrence of the disallowed G:G and G:A pairs at the wobble position. The chief
factor is, of course, the configuration of these two pairs, which represents a wide
divergence from the canonical Watson-Crick base pairs. It thus emerges from
this study that non-suitability of base pairing only at the wobble position can
itself be a sufficient criterion to rule out the disallowed near-cognate codonanticodon duplexes from occurring in nature during the translation process.
Table VI.3b (next page) lists values of the helical parameters for the four
duplexes studied here. Taking the GCC-GGC duplex as having the truly optimal
243
double-helical configuration, it is evident that, for most of the cases, a fair
approx-imation to the standard double-helical configuration is adhered to. The
helical angles d1 to d4 have normal values except for the d2 and d4 values in the
allowed GCC-GGU duplex. The e1, e3 and e3 distance parameters serve to
distinguish the disallowed duplexes from the allowed duplexes to some extent,
where the NON duplexes have e1 values larger or smaller than do the WC and
AW
duplexes.
244
The
Table VI.3b Helical parameters for trinucleotide duplexes arising from the GCC anticodon
________________________________________________________________________________________________
Duplex
Angles
_____________________________
Distances
____________________________
Dihedrals
________________
d1
d2
d3
d4
e1
e2
e3
e4
f1
f2
________________________________________________________________________________________________
GCC- GGA
91.20
49.24
110.33
47.12
5.775
5.636
5.843
5.506
-64.45
-68.42
GCC- GGG
108.36
43.02
111.96
48.00
5.452
5.949
5.635
5.547
-80.56
-70.26
GCC- GGU
94.96
74.11
95.69
70.02
5.515
5.556
5.218
5.221
-64.05
-50.17
GCC- GGC
108.50
51.78
112.52
48.14
5.496
5.422
5.504
5.456
-73.22
-71.54
________________________________________________________________________________________________
a
Angles in degrees; distances in angstrom
245
Table VI.3c Stacking interaction parameters for the four duplexes arising from the GCC anticodon
_______________________________________________________________________________________________
Duplex
Angles
____________________________
Distances
__________________________
Dihedrals
___________________
p1
p2
p3
p4
q1
q2
q3
q4
r1
r2
_______________________________________________________________________________________________
GCC- GGA
87.43 54.45 107.85 52.38
5.198 4.902
5.082
4.824
-59.13
-63.17
GCC- GGG
105.20 43.86 108.97 53.21
5.141 4.907
5.456
4.826
-77.52
-64.48
75.23
4.883 5.019
4.545
4.556
-64.13
-48.19
108.78 53.65
4.753 4.722
4.817
4.732
-67.93
-65.04
GCC- GGU
GCC- GGC
89.78
75.29
104.20 57.27
92.05
_______________________________________________________________________________________________
a
Angles in degrees; distances in angstrom
246
Table VI.3d Backbone torsional angles for duplexes arising from the GCC anticodon (with G as AWB).
__________________________________________________________________________________________________
Duplex
Individual
sequence
5' terminal
_______________________
Non terminal
_______________________
3' terminal
__________________
γ
δ
ε
γ
δ
ε
γ
δ
_________________________________________________________________________________________________
GCC-GGA
GCC-GGG
GCC-GGU
GCC-GGC
GCC
56.24
75.44
-159.84
57.77
75.94
-166.44
58.93
83.60
GGA
-169.96
76.60
-155.99
60.67
74.93
-160.33
60.71
77.92
GCC
-169.98
74.74
-160.28
57.61
73.45
-175.64
179.51
80.97
GGG
56.07
75.35
-163.65
58.94
74.73
-159.99
59.87
77.75
GCC
56.27
75.64
-164.81
61.86
77.06
-166.44
59.59
81.24
GGU
56.06
75.14
-169.57
60.77
80.02
-165.06
60.95
81.07
GCC
55.98
74.91
-161.37
59.96
73.89
-162.06
58.32
78.09
GGC
55.53
73.80
-162.35
59.67
75.20 -161.45
60.11
77.10
__________________________________________________________________________________________________
247
a
Angles in degrees; distances in angstrom
248
e2, e3 and e4 values are also all larger for the disallowed NON duplexes than for
the allowed WC and AW duplexes.
With regard to the stacking parameters listed in Table VI.3c (two pages
earlier), it is the distances q1, q3 and to a lesser extent q3 which more effectively
differ-entiate between the allowed and disallowed duplexes, where the
disallowed GCC-GGG duplex emerges as probably the most distorted on this
account. These distance parameters are noticeably larger for the NON duplexes
than for the WC and AW duplexes. One also notes that the allowed AW duplex
GCC-GGU has its angle parameters p2 and p4 uniquely larger than all the rest in
the series.
Table VI.3d (previous page) gives the torsional angle parameters related to the
backbone of the duplexes. Most of the values are about the same for all cases,
which points to the observation that, in general, modifications around the
wobble position do not affect these backbone torsional angles very much.
Exceptions are noted for some 5' terminal sequences, viz., the GGA sequence of
the disallowed GCC-GGA duplex and the GCC sequence of the disallowed GCCGGG duplex, where the γ angle parameter has a value of about -170° as
contrasted with the more usual value of about 56°. In this, the situation
resembles that around the G residue of Z-DNA. The 3' terminal side of the GCC
sequence of the disallowed GCC-GGG duplex is also associated with an
anomalous γ angle value of ≈ 180°.
VI.3.2 Duplexes with Uracil as AWB
The glycine anticodon UCC is made to pair with the codons GGA, GGG, GGU and
GGC, leading to four anticodon-codon duplexes. Only the UCC-GGA duplex is
allowed and cogante, being of the WC type, while the UCC-GGG, UCC-GGU and
249
UCC-GGC are all non-cognate and disallowed, being of the NON type. These 4
duplexes are schematically depicted in Figs. IV.6a to IV.6d (next two pages).
The tRNAs containing guanine as an AWB can easily read a uracil-ending codon
by wobbling, whereas those tRNAs containing uracil as an AWB read guanineending codons less efficiently. Analysis of tRNA genes from 50 genomes of
Eukarya, Archaea and Bacteria revealed that C34-containing tRNAs (C is AWB) are
almost universally used in eukaryotes to read G-ending codons like CAG, AAG or
GAG [73], leading to the conclusion that eukaryotes may not use
250
OH
H
N
HO H
3/
ANTICODO
N
H
H
N
O
C2
H
O
H
N
N2
H
O
P
O
O
H
HO H
H C3
H
N
N3
O
O
N
H
N
O
H
NH
H
N
N4
N
H OH
O
Fig. IV.6a The UCC-GGA duplex.
H
H
OH
H
C4
O
P
N
HO
251
O
O
H
C5
O
O
H
O
N
N5
NH
H
5/
-
N
N
H
O
P
O
H
O
O
H
HN
O
O
H OH
N
5/
-
H
H
C6
N
HO H
P
N
N6
O
H
O
O
H
O
O
HN
-
N
N
N
N1
H C1
HO
NH
H OH
CODON
O
O-
3/
O
OH
H C1
HO
ANTICOD
ON
H
HO H
P
O
H
N
O
O
C2
H
O
H
N
N2
O
H
O
H OH
HO H
H C3
O
H
N
N3
O
O
H
N
H
N
O
H
NH
O
P
H OH
O-
O
N
N
N4
N
O
O
-
H
H
C5
H
O
P
5/
O
H
O
N
N5
NH
H
O
P
O
H
O
O
O
N
N
H
HN
O
C6
N
5/
-
H
H2N
O
H
H
H
N
HN
-
N
H
O
N
N6
CODON
3/
N
O
N
N1
HO H
O
H
N
3/
H
OH
H
C4
H OH
HO
Fig. IV.6b The UCC-GGG duplex
OH
O
O
H
O
HO H
3/
H
N
N1
H
O
N
ANTICOD
O
O
H
N
HO H
P
C2
H
O
H
N
N2
O
P
O
H
O
HO H
H C3
H
N
N
N3
O
HO
252
H
N
N4
N
H
NH
H OH
O
H
H
OH
H
C4
O
P
N
N
O
H
O
H
C5
O
O
H
O
N
N5
NH
H
O
-
N
N
H
5/
O
O
H
O
O
H
HN
O
H OH
N
5/
-
C6
O
P
O
H
O
O
O
H
O
HN
-
N
N6
H C1
HO
H
N
H OH
CODON
H
N
O-
3/
Fig. IV.6c The UCC-GGU duplex
OH
N
HO H
3/
H C1
HO
ANTICOD
ON
H
N
N1
O
H
N
HO H
P
C2
H
O
H
N
N2
H
O
P
O
C6
O
N
N5
N
H
HO H
H C3
H
O
N
N3
O-
O
H
O
N
H
N
O
H
NH
H
O
H
C5
NH
H
5/
N
N
O
O
P
H OH
O
N
N
N4
N
O
P
H OH
O
O
H
O
H
HN
O
O
H
O
5/
-
N
N6
O
H
O
H
O
O
H
O
N
O
HN
-
H
NH
H
H
CODON
H
O
O-
3/
OH
H
C4
H OH
HO
Fig. IV.6d The UCC-GGC duplex
U-G wobbling [74,75]. It is believed though that efficient U:G wobbling strongly
depends on enzyme mediated C5-modifications of the uracil ring [75-78]. Whenever U34 is not post-transcriptionally modified, it reads all the four synonymous
codons by processes like ‘two-out-of-three decoding’ [79], ‘4-way wobbling’ [80]
or ‘superwobbling’ [81]. Such undiscriminating codon reading by the unmodified
U34 depends on the identity of the other two bases at position 35 and 36 as well
as on the extra-anticodonic base sequences like a cytosine in position 32 (C 32) of
the anticodon loop [82]. Such a decoding system is used only in Bacteria, but
never in Archaea or in Eukarya [70], to read codons like Val-GUN3, Pro-CCN3 and
Ala-GCN3, less frequently for Leu-CUN3, Ser-UCN3, Thr-ACN3 and exceptionally for
Gly-GGN3 (N stands for any of the four major RNA bases at the third position).
253
Table VI.4a (next page) presents the interacting edges, pairing energies and
configuration data of the individual base pairs present in these 4 anticodoncodon duplexes in which an unmodified uracil residue occupies the AWP. All the
three bases of the WC duplex UCC-GGA are standard Watson-Crick base pairs,
with Rcc values from 10.68 to 10.75, 1 and 2 values from 54.10 to 57.85, and
dihedral c values from 0.09 to 9.67. The pairing energy Ep for this WC duplex is 36.26 kcal mol-1. Note that this duplex UCC-GGA is less stable than the WC
duplex GCC-GGC (see Sec. VI.3.1) because of the presence of the A:U pair in the
former which forms two H-bonds, while all the G:C or C:G pairs in the latter form
three H-bonds each.
The disallowed duplex UCC-GGG contains a U:G pair at the wobble position
while the other two pairs are Watson-Crick base pairs. The Rcc value for the U:G
pair is 10.55, 1 and 2 values are 70.78 and 43.13, and the dihedral c is 0.57°.
The Ep value of this duplex is -31.19 kcal mol-1. The stability of this duplex
containing the wobble pair U:G is lesser than the allowed AW duplex GCC-GGU
(previous section) that contains a G:U wobble pair by only 1 kcal mol-1. Even so,
the duplex UCC-GGG never occurs in eukaryotes [64, 65]. Just as the solitary U:G
and G:U pairs have been difficult to differentiate computationally in the earlier
Chapters of this Dissertation, so here too it is difficult to differentiate between
these two pairs in the context of trinucleotide duplexes.
Table VI.4a Configurational dataa of anticodon codon pairs with uracil as AWB
after energy minimization by xmin (solvated and neutralised with Na+ ions).
__________________________________________________________________
Base
Edges
Ep
Rcc
θ1
θ2
c
Pair
__________________________________________________________________
Duplex
UCC-GGA
(Obsd)
U:A (I) wc/wc
C:G (II) wc/wc
C:G (III) wc/wc
-31.26
10.68
10.75
10.67
55.54
56.40
57.85
57.41
54.10
54.44
0.09
8.99
9.67
UCC-GGG
(Unobsd)
U:G (I)
C:G (II)
-31.19
10.55
10.73
70.78
56.84
43.13
53.32
0.57
9.62
wc/wc
wc/wc
254
C:G (III) wc/wc
UCC-GGC
(Unobsd)
U:C (I) wc/wc
C:G (II) wc/wc
C:G (III) wc/wc
-32.03
10.69
57.38
54.37
10.31
8.61
10.68
10.71
59.90
57.13
57.26
61.97
53.58
54.90
10.50
3.26
3.01
UCC-GGU
(Unobsd)
U:U (I) wc/wc -31.04
8.67
80.91
44.80
9.51
C:G (II) wc/wc
10.65
57.60
54.27
3.52
C:G (III) wc/wc
10.74
56.72
54.50
2.61
__________________________________________________________________
a
Pairing energies in kcal/mol; distances in angstrom; angles in degrees
The two NON type duplexes UCC-GGC and UCC-GGU (both disallowed) contain
the narrow pyrimidine-pyrimidine base pairs U:C and U:U at their wobble
positions. Their pairing energy Ep values are -32.03 and -31.04 kcal mol-1 respectively, which are more or less about the same as for the allowed UCC-GGA duplex.
The Rcc values of these small U:C and U:U pairs are 8.61 and 8.67 Å, the dihedral
c values are 10.50 and 9.51, while the 1 and 2 values range from 44.80 to
80.91. Thus, the values of the configuration markers deviate quite a bit from the
standard Watson-Crick alignments, especially with regard to the small Rcc
distances, thus preventing these two NON type duplexes from occurring at the
wobble position.
The three-dimensional structures of these four anticodon-codon duplexes
arising from the glycine UCC anticodon as obtained from these AMBER
calculations are represented in Fig. VI.7 (next page), with the atoms given as
points and the bonds drawn as sticks of appropriate colour.
255
UCC-GGA
UCC-GGC
256
UCC-GGG
UCC-GGU
Fig. VI.7 Three-dimensional view of optimized duplexes arising from UCC anticodon
Table VI.4b Helical parameters for anticodon-codon duplexes arising out of the UCC anticodon for glycine
__________________________________________________________________________________________________
Duplex
Angles
___________________________
Distances
___________________________
257
Dihedrals
_____________________
d1
d2
d3
d4
e1
e2
e3
e4
f1
f2
__________________________________________________________________________________________________
UCC- GGA
113.58
51.09
113.95 47.03
5.450
5.459
5.491
5.506
-67.26
-69.71
UCC- GGG
123.56
47.76
116.49 45.01
5.301
5.624
5.518
5.587
-64.90
-73.39
UCC- GGC
125.33
60.82
112.57
56.17
5.466
5.669
5.236
5.323
-75.11
-57.74
UCC- GGU
137.27
57.51
118.15 49.44
5.420
5.245
5.304
5.514
-59.42
-66.78
_________________________________________________________________________________________________
a
Angles in degrees; distances in angstrom
Table VI.4c Stacking interaction parameters for anticodon-codon duplexes arising out of the UCC anticodon for glycine.
__________________________________________________________________________________________________
Duplex
Angles
_______________________________
Distances
____________________________
258
Dihedrals
__________________
p1
p2
p3
p4
q1
q2
q3
q4
r1
r2
__________________________________________________________________________________________________
UCC- GGA
111.06
54.83
110.82
52.08
4.726 4.891
4.908
4.813
-62.71
-63.40
UCC- GGG
120.42
53.62
113.28
50.31
4.420
4.913
4.847
4.902
-54.52
-65.86
UCC- GGC
122.07
65.37
112.57
56.17
4.655 4.933
4.641
4.617
-69.85
-53.34
UCC- GGU
132.45
64.63
118.15
49.44
4.474 4.526
4.672
4.872
-45.98
-60.50
__________________________________________________________________________________________________
a
Angles in degrees; distances in angstrom
TABLE VI.4d Backbone torsional angles for anticodon-codon duplexes arising out of the UCC anticodon for glycine
_________________________________________________________________________________________________
Individual
5' terminal
Non terminal
259
3' terminal
Duplex
sequence
_______________________
_________________________
_________________
γ
δ
ε
γ
δ
ε
γ
δ
_________________________________________________________________________________________________
UCC- GGA
UCC- GGG
UCC- GGC
UCC- GGU
UCC
56.01
74.84
-161.24
60.06
74.43
-161.85
59.01
80.35
GGA
56.00
75.37
-161.76
62.15
75.07
-161.53
61.07
77.12
UCC
55.97
74.93
-160.78
61.46
75.99
-158.24
59.31
77.46
GGG
56.85
77.30
-156.77
60.77
75.66
-159.91
60.58
77.30
UCC
56.08
75.07
-164.24
63.57
77.66
-157.84
61.62
78.50
GGC
56.53
75.83
-160.10
59.28
76.41
-163.19
61.03
76.23
UCC
55.85
74.71
-162.46
63.25
78.91
-155.19
GGU
55.67
74.27
-161.80
61.33
75.45
-162.46
59.87
62.06
76.73
76.32
_________________________________________________________________________________________________
a
Angles in degrees; distances in angstrom
260
Tables IV.4b, IV.4c and IV.4d respectively present the helical parameters, the
stacking parameters and the backbone torsional angles for the anticodon-codon
duplexes arising out of UCC as anticodon. Among the various helical parameters,
d1 has values larger for the disallowed duplexes (especially the UCC-GGU pair).
Among the stacking parameters, p1 and p3 are smaller for the allowed WC duplex
UCC-GGA, while q1 and q3 are larger for this allowed duplex. The backbone
torsional angles do not reveal significant differences between the allowed and the
disallowed duplexes, except that, for the allowed duplex at the 3' terminus, the γ
angle is the smallest and the δ angle is the largest.
VI.3.3 Duplexes with Cytosine as AWB
The glycine anticodon CCC is made to pair with the codons GGA, GGG, GGU and
GGC, leading to four anticodon-codon duplexes. Only the CCC-GGG duplex is
allowed and observed, being of the WC type, while the CCC-GGA, CCC-GGU and
CCC-GGC are all non-cognate and disallowed, being of the NON type. Note that
transfer RNAs harboring an unmodified cytosine residues at the AWP are very
restrictive and read codons ending only with guanine residues.
Table VI.5a (next page) presents the interacting edges, pairing energies and
individual base pair configuration data of anticodon-codon duplexes in which a
cytosine residue occupies the AWP (as depicted in Figs. IV.7a to IV.7d, next and
in the two succeeding pages). All three bases of the allowed WC duplex CCC-GGG
are standard Watson-Crick base pairs whose Rcc values range from 10.68 to
10.72 Å, 1 and 2 values from 53.74 to 57.87, and c values from 0.58 to 9.14°.
The Ep value for this duplex is quite substantial, being-36.44 kcal mol-1.
The NON type duplex CCC-GGA contains a C:A base pair at the wobble position
which forms only one true H-bond, as reflected in the low stability of the duplex
with its Ep value of only -27.85 kcal mol-1. The Rcc value is 11.02, the c value is
19.19° and the 1 and 2 values are 60.08 and 39.22° respectively. Thus, the
values of the configuration markers deviate quite a bit from the standard
261
Watson-Crick alignments for this base pair. All these factors indicate that this C:A
base pair is not suitable to be accommodated at the wobble position.
Table VI.5a Configuration dataa of anticodon-codon pairs with cytosine as AWB
after energy minimization by xmin (solvated and neutralised with Na+ ions)
__________________________________________________________________
Duplex
Pair
Edges
Ep
Rcc
θ1
θ2
c
__________________________________________________________________
CCC-GGA
(Unobsd)
C:A (I)
wc/wc
C:G (II) wc/wc
C:G (III) wc/wc
-27.85
11.02
10.78
10.61
60.08
56.44
58.53
39.22
52.31
55.44
19.19
12.17
3.45
CCC-GGG
(Obsd)
C:G (I) wc/wc
C:G (II) wc/wc
C:G (III) wc/wc
-36.44
10.68
10.72
10.69
57.87
56.85
57.56
53.74
53.95
54.42
0.58
8.29
9.14
CCC-GGU
(Unobsd)
C:U (I)
wc/wc
C:G (II) wc/wc
C:G (III) wc/wc
-32.71
8.72
10.66
10.72
63.56
57.58
57.16
56.41
54.14
54.81
11.42
1.36
0.55
CCC-GGC
(Unobsd)
C:C (I)
wc/wc -29.27
9.34
68.80
40.00
12.20
C:G (II) wc/wc
10.71
56.95
53.23
25.88
C:G (III) wc/wc
10.49
56.33 56.70
21.00
__________________________________________________________________
a
Pairing energies in kcal/mol; distances in angstrom; angles in degrees
262
NH2
OH
HO H
3/
O
H
HO H
P
O
H
N
O
O
O
N
N2
C2
H
H
O
H
NH
H
HO H
P
N
O
N
N3
H C3
O
H
O
O
H OH
N
N4
N
H
O
P
O-
O
N
N
O
H
H
O-
H
H
C5
H
O
O
O
H
O
H
5/
O
O
N
N5
N
H
HN
O
H OH
N
O
O
P
N
5/
-
O
O
H
HN
-
C6
H
H
H
N
H C1
H
O
N
N6
N
HO
ANTICO
DON
N
CODON
N
N1
NH
H
N
3/
H
OH
H
C4
H OH
NH
HO
Fig. IV.7a The CCC-GGA duplex
OH
H C1
HO
ANTICOD
ON
H
N
N1
O
H
O
HO H
C2
H
O
H
N
N2
O
P
O
HO H
H C3
H
N
N3
O
HO
263
O-
O
H
O
N
H
N
O
H
NH
H
O
H
C5
O
N
N
N4
N
H
H
OH
H
C4
O
P
H OH
O
O
H
O
N
N5
NH
H
5/
N
N
H
O
P
H OH
O
H
O
O
H
HN
O
C6
N
5/
-
O
H
NH
H
N
P
H
O
H
O
O
H
O
N
N6
N
N
HN
-
N
O
H
N
HO H
3/
H
H OH
CODON
HN
O-
3/
Fig. IV.7b The CCC-GGG duplex
OH
H
O
N
HO H
3/
H C1
HO
ANTICOD
ON
H
N
N1
O
C2
H
O
H
N
N2
O
O
O
H
HO H
H C3
H
N
N3
O
C5
O
N
H
N
O
H
NH
H
N
N4
N
O
Fig. IV.7d The CCC-GGU duplex
H
H
OH
H
C4
O
P
H OH
N
HO
264
O
O
H
O
O
H
O
N
N5
NH
H
5/
-
N
N
H
O
P
H OH
O
H
P
O
C6
O
H
HN
O
O
H
N
5/
-
O
H
N
HO H
P
N
N6
H
O
H
O
O
H
O
N
O
HN
-
H
H OH
CODON
HN
O-
3/
OH
NH2
H
N
N
N1
HO H
3/
ANTICOD
ON
H
O
HO H
P
O
H
N
O
O
C2
H
O
H
N
N2
O
H
O
P
O
C6
O
O
H
HO H
H C3
H
O
N
N3
O
C5
O
N
H
N
O
H
NH
H
O
H
O
N
N
N4
N
O
P
H OH
O
O
H
O
N
N5
NH
H
5/
-
N
N
H
O
P
H OH
N
HN
O
O
H
O
H
5/
-
H
O
H
HN
-
N
N6
O
H C1
HO
H
O
N
H
H
CODON
H
N
O-
3/
OH
H
C4
H OH
HO
Fig. IV.7c The CCC-GGC duplex
The NON type duplexes CCC-GGU and CCC-GGC with Ep values of -32.71 and 29.27 kcal mol-1 respectively contain the pyrimidine-pyrimidine base pairs C:U
and C:C at their respective wobble positions. The Rcc values of the C:U and C:C
wobble pairs are 8.72 and 9.34 Å respectively, the c values are 11.42 and
12.20°, while the 1 and 2 values range from 40.00 to 68.80. The C:C pair also
has only one true H-bond and shows a lower stability. Thus it is the short Rcc
values and the weakness of H-bonding at the wobble position that prevent these
two NON type duplexes from occurring during codon-anticodon pairing in
nature.
Fig. IV.8 (next page) portrays the three-dimensional fully optimized structures
of the four anticodon-codon pairs arising out of the glycine CCC anticodon,
265
where the mini-helices are evident and the central base pair is present in a
sidelong view. Atoms are given as points and the bonds (including the hydrogen
bonds) are shown in colour code.
266
CCC-GGA
CCC-GGC
267
CCC-GGG
CCC-GGU
Fig. VI.8 Three-dimensional view of optimized duplexes arising from the CCC anticodon
268
Table VI.5b Helical parameters for anticodon-codon duplexes arising out of the CCC anticodon for glycine
_________________________________________________________________________________________________
Duplex
Angles
______________________________
Distances
____________________________
Dihedrals
_________________
d1
d2
d3
d4
e1
e2
e3
e4
f1
f2
_________________________________________________________________________________________________
CCC- GGA
112.89
55.85
111.11
54.74
5.443
5.732
5.588
5.444
-51.16
-53.77
CCC- GGG
116.74
47.97
115.70
46.22
5.493
5.572
5.542
5.558
-68.32
-71.17
CCC- GGU
127.52
59.90
115.01
53.70
5.429
5.430
5.291
5.394
-70.48
-59.63
CCC- GGC
118.28
71.97
105.49
66.73
5.436
5.393
5.311
5.403
-36.53
-36.61
_________________________________________________________________________________________________
a
Angles in degrees; distances in angstrom
269
Table VI.5c Stacking interaction parameters for anticodon-codon duplexes arising out of the CCC anticodon for glycine
_______________________________________________________________________________________________
Duplex
Angles
_____________________________
Distances
___________________________
Dihedrals
_________________
p1
p2
p3
p4
q1
q2
q3
q4
r1
r2
_______________________________________________________________________________________________
CCC- GGA
112.69
58.99
110.61
57.48
4.795
5.018
4.887
4.831
-45.39
-51.02
CCC- GGG
114.64
52.17
112.59
51.22
4.753
4.961
4.867
4.882
-62.59
-64.39
CCC- GGU
124.28
65.44
112.42
57.23
4.655
4.667
4.670
4.742
-63.43
-54.86
CCC- GGC
117.95
74.94
105.96
66.98
4.799
4.615
4.708
4.862
-30.72
-35.55
_______________________________________________________________________________________________
a
Angles in degrees; distances in angstrom
270
Table VI.5d Backbone torsional angles for anticodon-codon duplexes arising out of the CCC anticodon for glycine
______________________________________________________________________________________________
Duplex
Individual
sequence
5' terminal
_______________________
Non terminal
________________________
3' terminal
_______________
γ
δ
ε
γ
δ
ε
γ
δ
______________________________________________________________________________________________
CCC- GGA
CCC- GGG
CCC- GGC
CCC- GGU
CCC
55.90
74.95
-162.71
59.20
75.70
-163.50
61.68
82.25
GGA
56.99
77.37
-154.90
61.83
74.76
-162.34
60.78
77.93
CCC
55.95
74.87
-161.06
60.58
74.61
-161.12
58.88
76.99
GGG
56.25
75.87
-159.75
61.66
75.01
-160.84
60.80
77.18
CCC
56.11
75.22
-166.03
63.65
77.58
-164.83
62.01
83.79
GGC
56.09
74.45
-159.12
62.22
75.52
-165.89
61.67
79.07
CCC
55.84
74.83
-163.61
62.63
75.87
-160.75
60.53
76.12
GGU
55.71
75.05
-158.13
61.56
75.49
-163.72
61.95
76.14
________________________________________________________________________________________________
271
a
Angles in degrees; distances in angstrom
272
The helical parameters, stacking parameters and backbone torsional angles for the
anticodon-codon duplexes arising out of the glycine anticodon CCC are given in
Tables VI.5b, VI.5c and VI.5d respectively (previous three pages).
From the helical parameters of Table VI.5b, it may be seen that, among the four
CCC-bases duplexes, the allowed WC duplex CCC-GGG has the smallest values of the
d2 and d4 angles, the largest of the e1 and e4 distances, and the largest f2 angle. The
two NON duplexes with pyrimidines at the wobble position appear more distorted in
relation to the WC structure. The NON duplex CCC-GGU has the largest d1 angle
value, the smallest e1, e3 and e4 distance values and the largest f1 dihedral value.
The NON duplex CCC-GGC has the largest d2 and d4 values, the smallest d3 value,
the smallest e2 value and the smallest f1 and f2 values. The NON duplex CCC-GGA
with the purine A at the wobble position has less distortion, with the largest e2 and
e3 values and the smallest d1 value.
The stacking parameters of Table VI.5c show that the WC duplex CCC-GGG is
associated with the smallest p2 and p4 angle values, the largest q4 distance value,
and the largest r2 value. The two NON duplexes with pyrimidines at the wobble
position demonstrate appreciable distortion from this optimal WC alignment. The
NON duplex CCC-GGU has the largest p1 value, the smallest q1, q3 and q4 values,
and the largest r1 value. The NON duplex CCC-GGC is also distorted with relation to
the WC duplex, with the largest p2 and p4 values, the largest q1 value, the smallest
q2 value and the smallest r1 and r2 values. The NON duplex CCC-GGA is less
distorted, and closer to the WC arrangement, with intermediary values of the
stacking parameters, except that it has the largest q2 and q3 values.
The backbone torsional angle data of Table VI.5d does not reveal significant
differences between the various allowed and disallowed duplexes, showing that the
backbone shape is not much affected by the nature of the base pair at the wobble
position of these anticodon-codon duplexes. It thus emerges that it is the helical and
the stacking parameters which are able to distinguish the disallowed duplexes from
the allowed WC duplex CCC-GGG. Here, it is the duplexes with pyrimidines at the
273
codon wobble position that display maximal distortion, while the duplex with the
wobble pair C:A occupies an intermediary position.
VI.4 Conclusions
The following conclusions emerge from this AMBER computational study on codonanticodon pairing involving the three glycine anticodons GCC, UCC and CCC which do
not have any minor bases:
1. The results of xmin classical molecular mechanical geometry optimization
method clearly indicates the stability order among these three kinds of duplexes is
WC type > AW type > NON type.
2. Likewise, the degree of distortion from the optimal duplex alignment gives the
general order NON type > AW type > WC type.
3. Configuration of the wobble base pair in the context of the full anticodon-codon
duplex still serves the role of principle discriminator between the allowed and the
disallowed duplexes, and in this it follows the key role demonstrated by
configuration of the solitary wobble pair as studied in the previous Chapters.
4. It is the helical and stacking parameters, rather than the backbone torsional
angles, which act as indicators of departure of duplex geometry from the standard
WC alignment of the anticodon-codon duplex.
274
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