Proc. Int. Symp. Biomol. Struct. Interactions, Suppl. J. Biosci.,
Vol. 8, Nos 3 & 4, August 1985, pp. 823–835. © Printed in India.
Conformational studies on nucleotide–amino acid interactions
leading to origin of life
R. BALASUBRAMANIAN
Department of Crystallography and Biophysics,* University of Madras, Guindy Campus,
Madras 600 025, India
Abstract. Living processes may be defined as the self-sustained chemical reactions based on
the special chemical machinery of nucleic acid-directed protein synthesis. Its genesis may be
traced to the molecular interaction between nucleotides and amino acids leading to a primitive
adaptor-mediated ordered synthesis of polypeptides. A primitive decoding system is described
and its characteristics are shown to imitate, in a primitive manner, the present-day elaborate
machinery of protein synthesis. This molecular interaction theory may be rightly considered as
the missing link between the Protochemical and Biological Evolution. The origin of chiral
specificity observed in living organisms is also traced to this specific molecular interaction in
the protobiological milieu.
Keywords. Chicken-and-egg problem; primitive decoding apparatus; Proto-tRNA; origin of
genetic code; chiral specificity; primitive protein synthesis; link between protochemical and
biological evolution; definition of living organisms.
Introduction
A study of origin of life necessarily involves the question of what is the underlying
principle behind the self-sustained chemical processes that go on in living organisms.
These chemical reactions are catalyzed and coordinated by proteins. These chain
molecules of amino acid residues are in turn synthesized by a complex machinery
involving nucleic acids, and so-produced proteins themselves. Thus the origin of this
protein synthesizing machinery poses a chicken-and-egg problem.
Moreover, this protein synthesizing mechanism has a peculiar characteristic. The
amino acids are not directly and simply condensed into polypeptides through catalysis.
An adaptor molecule tRNA, charged with a specific amino acid at its one end,
corresponding to a triplet of nucleotides (anticodons) situated at another end, plays a
crucial role in a complex process. The anticodon triplet of nucleotide “recognizes” a
triplet codon through Watson-Crick base pairing on another long chain nucleic acid
namely mRNA. The consecutive triplet codons on mRNA are recognized by
appropriate tRNAs having complementary anticodons and the amino acids charged to
these tRNAs condense into ordered polypeptide sequences. Thus protein synthesis in
biological organisms happens to be a nucleic acid-directed, adaptor mediated, ordered
synthesis. It may be noted that this nucleic acid-directed protein synthesis is found in all
living organisms and is conspicuous by its absence in all non-living organization of
* Contribution No. 665 from this department.
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Balasubramanian
matter. Also, since this forms the basis of all biological functions this may be rightly
taken for a definition of life.
Even with the minimum number of necessary and sufficient components, viz.,
mRNA, tRNA and charging enzymes synthetases, this molecular machinery is so
complex that it could not have arisen all at once (lock stock and barrel) in the prebiotic
milieu. Moreover in any stochastic theory for origin of life, the probability for the
establishment of a particular machinery like this is extremely small. Hence one has to
look for a simple molecular interaction that could have evolved and culminated into the
present-day precision mechanism. Several possible molecular associations have been
discussed in the literature (see Hopfield, 1978; Balasubramanian, 1980;
Balasubramanian et al., 1980; Hartman, 1984), but none of them seems to give a precise
molecular interaction rationale for the origin of this mechanism.
Origin of the primitive decoding system
We considered a pentanucleotide having uracil at the 5'-end, a purine at the 3'-end
flanking any three bases in the middle (Balasubramanian, 1979; Balasubramanian et al.,
1980). One of the most favourable conformations of this oligonucleotide is the U-turn
conformation in which there is a strong hydrogen bond between the donor N3-H3 of
uracil and highly electronegative oxygen of the fourth phosphate, with the second, third
and fourth bases stacked into an RNA-11 helical conformation. These latter three
residues are termed as primitive anticodons (PAC) for reasons that follow. This
conformation has a ‘cleft’ formed between U(l)* and PAC. An amino acid was nestled
into this cleft making use of some specific hydrogen bonds. The 3'-end purine of the
pentanucleotide was also made use of for this cosy nestling.
This hydrogen-bonding molecular interaction between a pentanucleotide and an
amino acid at once turned out to be a primitive decoding system, in which the
pentanucleotide serves as a primitive tRNA (PIT) of a double-sided template, wherein
PAC is capable of base pairing with a codon sequence on another long chain RNA
(primitive mRNA, PIM) while PAC’s other side can discriminate amino acid residues,
depending upon the base sequence.
The specific hydrogen bonds that hold the amino acid in the PIT (figure 1) are:
(i) One of the carboxyl oxygen of the amino acid accepts a proton from the 2'hydroxyl of U(l) ⎯AAHB1.
(ii) The amino group of amino acid donates one proton to C2–O2 of base U(l)⎯
AAHB2.
(iii) This amino group donates another proton to N7 of base A(5)—AAHB3.
These three specific hydrogen bonds constrain the amino acid to have a specific
configuration in which the R-group of amino acid points towards a surface of PAC,
called the backside-surface (another front-side surface is free to interact with codons on
PIM, figure 2). The interactive characteristics of this backside surface of PAC changes
* Throughout the text, numbers within parenthesis (like U(l)) refer to the residue number in the
pentanucleotide.
Nucleotide-amino acid interactions
825
Figure 1. Conformation of Primitive tRNA(PIT) holding an amino acid through three
hydrogen bonds AAHB1, AAHB2, AAHB3. The U-turn hydrogen bond N3–H3 – – – OL is
seen in the middle of the diagram. There is another hydrogen bond O2'– –H2 of G(3) – – – O1
of C(4) which constrain the bases G(3) and C(4) to have a standard RNA-11 conformation.
Such a hydrogen bond is absent between sugars (2) and (3). The residues in the pentanucleotide
are numbered from the 5'-uracil and these residue numbers are shown within parenthesis.
with the sequences of bases in PAC and we show below specific correlations
between the PAC sequences and the R-groups of amino acids for favourable
interactions; and this correlation corresponds to genetic code. Thus our molecular
interaction theory at once gives a rationale for the origin of a decoding system and also
for the origin of the genetic code (see also Balasubramanian, 1981, 1982).
The genetic code
Let us consider a few key amino acids from the codon table and the corresponding
anticodon sequences (Balasubramanian and Raghunathan, 1984). Figure 3 shows
Threonine nestled in a PIT having PAC sequence 5'-GGU-3'. It may be seen that OG2H of Thr gets locked up into a good hydrogen bond with O4 of U(4). This hydrogen
bond uniquely fixes the orientations of the other groups attached to CB, viz., HB and
the methyl group CG1 (H3). The methyl group has interactive contact with the base
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Balasubramanian
Figure 2. The “inside” or the backside and the “outside” or front-side of the PAC are shown
schematically over a suitable projection of the molecular moiety. The frontside of PAC is in
such a configuration as to readily base-pair with a codon-sequence while the backside
molecular surface can specifically interact with side-chain of an amino acid residue. The
C L E F T formed by the U-turn conformation and the amino acid nestled therein are
schematically shown for a clear comprehension of the molecular mechanism of the primitive
decoding system.
G(3) providing a close packed conformation for molecular association of this PIT and
Thr.
Energy calculations were carried out by varying the side chain dihedral angles χ1, χ21,
χ22 of Thr. Since many of the conformations were eliminated by short contacts we
could start at a reasonable conformation, vary one dihedral angle at a time and cyclize
this procedure to arrive at the minimum energy conformation of χ1= χ21= – 40°
and χ22 = – 90°. The parameters of the hydrogen bond OG2-HG2– – – O4 (of U(4))
are: Η – – – Ο = 1·8 Å and angle Ο—Η – – – Ο = 155°.
The β-carbon of Thr is asymmetric and on interchanging any two groups attached to
CB we get allothreonine. When we tried to nestle allothreonine in this PIT, the
interaction became unfavourable due to loss of the hydrogen bond and due to short
contacts between its R-group and PAC bases, particularly G(3). Thus our molecular
interaction theory presents a new angle of view and interesting possibilities for the
elimination of non-proteinous amino acids in the very early stages of the development
Nucleotide-amino acid interactions
827
Figure 3. A suitable projection of the complex between PIT 5'-UGGUA-3' and LThreonine. The key atoms of interactions are marked. For further discussions see text.
of decoding system (for other explanations see Rohlfing and Saunders, 1978; Weber
and Miller, 1981).
When Gln is nestled in PIT having PAC sequence 5'-UUG-3', two hydrogen bonding
interactions are possible, between R-group of Gln and U(3) and G(4) of PAC.
NE1—Hl (Gln) – – – O4 of U(3)
NE1—H2(Gln) – – – O6 of G(4)
On replacement of bases U(3) and G(4) by other bases either short contacts develop or
hydrogen bonds are lost.
For the sister amino acid Asn in association with PAC, 5'-GUU-3', the following two
hydrogen bonds are formed
ND1—H1 (Asn) – – –O4 of U(3)
ND1—H2 (Asn) – – – O4 of U(4).
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Balasubramanian
Comparing the hydrogen bonding interactions pertaining to these two sister amino
acids, it is seen that the longer side-chain of glutamine is accommodated by the farther
carbonyl O6 of G(3) while the shorter side chain of Asn is interacting with the nearer
carbonyl O4 of U(3).
Thus the last two bases of PAC seem to be uniquely suited for these sister amino
acids Gln and Asn though the bases in the first (wobble) positions of ΡACs do not seem
to play any specific role.
Actually this is the case with most of the amino acids as the wobble anticodon is nonspecific for amino acids. In the codon table one can readily appreciate the fact that
though a doublet code takes care of most of the amino acids, a triplet frame for a
comma-free reading seems to be necessary. In our postulate there is a structural
explanation for such a situation. In the PIT, U-turn hydrogen bonded conformation is
an important feature for forming a cleft to nestle an amino acid. In this conformation
U(1) is locked up by a strong hydrogen bond with the fourth phosphate allowing a
trinucleotide (PAC) to remain in a regular helical conformation for possible
anticodonic function. Thus triplet frame of reading the code is a natural consequence of
this type of molecular association. The wobble behaviour of the first anticodon also
seems to be a natural consequence of the U-turn formation. This conformation actually
precludes another hydrogen bond between the first two sugars of the anticodon triplet,
allowing the first base to wobble, while it facilitates a hydrogen bond between the
second and third sugars of PAC, constraining these bases for a standard base-pairing
with the codon. This gives a structural explanation for the degeneracy in the code and
explains why an RNA happens to be the adaptor for protein synthesis.
(Balasubramanian and Seetharamulu, 1980, see also 1983).
Cystein is a sulphur containing amino acid having the anticodon sequence 5'-GCA3'. Our calculations show that the side chain-PAC interaction is characterised by two
hydrogen bonds
N6—H6 of A(4) – – – SG of Cys
SG—Η of Cys – – – N7 of A(4).
The second and third position of PAC seem to be uniquely fixed by considerations of
hydrogen bonding and steric possibilities. When we considered A in the second and C in
the third position of PAC, two hydrogen bonds were feasible, but the amino acid
conformation has an intra-amino acid short contact since sulphur has to be in cis
position with respect to carboxyl oxygen.
Methionine is an amino acid which has a non-degenerate single code and
interestingly its lengthy and bulky (sulphur at δ position) side chain reaches out
specifically to interact with the first position of PAC and there are two possible
hydrogen bonds
N4—H4 of A(3) – – – SD of Met
N6—H6 of C(2) – – – SD of Met.
Interaction energies of all the other amino acids and their cognate anticodons have also
been studied critically and they would be reported elsewhere.
Nucleotide-amino acid interactions
829
Primitive protein synthesis
Another interesting and important aspect of our theory is that when two PITs holding
their cognate amino acids, happen to base-pair with consecutive triplets on PIM, there
is a possibility of dynamic interaction by which, the amino acid held by PIT1 is brought
to proper juxtaposition of the amino acid held by PIT2, facilitating the formation of
peptide bond. Sequential happening of such events can lead to a primitive but ordered
synthesis of proteins in accordance with the sequence of bases (hence codons) on PIM.
Thus this molecular interaction neatly mimicks the present-day machinery of nucleic
acid-directed adaptor-mediated ordered synthesis of proteins, though in a miniature
form. This is probably the ‘logic’ behind the later evolution of present-day sophisticated
machinery. This is perhaps the rationale for the origin of this peculiar coupling process
between nucleic acids and proteins forming the basis of self-sustained biochemical
processes found in all living organisms.
A schematic diagram of this primitive decoding apparatus is shown in figures 4 and 5.
As a first step in substantiating the possibility of such molecular events, the essential
features for their stereochemical feasibility were simulated using a computer
(Balasubramanian and Seetharamulu, 1984). A projection diagram of the ‘final’
conformation in which the CO group of amino acid1 of PIT1 is brought to
Figure 4. Schematic diagram of PIT1 holding amino acid AA1 while PAC coupling with its
complementary codon in PIM. The adjacent codon is coupled to PIT2 carrying AA2. A
dynamic interaction is triggered and AA1 is brought to juxtaposition to AA2 as shown in
figure 5.
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Balasubramanian
Figure 5. In the final conformation of the dynamic interaction, AA1 and AA2 are shown
schematically to be in proper juxtaposition for formation of peptide bond.
juxtaposition to NH group of amino acid2 of PIT2 is shown in figure 6. The relevant
atoms Ο and Η are brought to hydrogen bonding distance of 2 A. Auto-condensation
of amino acids (without enzymes) is possible if amino acid esters are brought to
juxtaposition as elucidated by the experimental works of Weber and Orgel (1978),
Fukuda et al. (1981), Folda et al. (1982), and Mullins et al. (1984). In order to see
whether adenylated esters of amino acids could be accommodated into our proposed
mechanism, we did generate the molecular moiety (figure 6) and found that there is no
steric hindrance due to the additional molecular moiety.
Weber and Orgel (1978) envisage that for primitive protein synthesis, the amine
group of one amino acid ester might have been constrained to lie in just the correct
position to attack the ester group of another, or they could be attached to tRNA-like
sequences which, through their specific tertiary structure held the amino acids together
in the correct orientation, and this is precisely what emerges from our molecular
interaction theory. Moreover in this interaction, the amino acids are brought to
juxtaposition in an orderly and sequential way through adaptor molecules adapting
themselves to consecutive triplet codons on long RNA (PIM).
The link between protochemical and biological evolution
Thus at one stage of prebiotic chemical evolution when nucleic acids, and amino acid
esters were available in a primitive milieu, the above type of molecular associations
among them would naturally lead to a primitive nucleic acid-directed adaptormediated ordered synthesis of polypeptides and when such ordered polypeptide
Nucleotide-amino acid interactions
831
Figure 6. The final conformation shown schematically in figure 5 is shown here as an actual
molecular projection generated in a computer. Adenylate esters of amino acids are shown
projected, since such esters are known to undergo autocondensation into peptides without the
aid of enzymes.
sequences happen to be those corresponding to crude enzymes like synthetases,
polymerases and replicases, biochemical processes would become, viable and selfsustaining, to evolve into living organisms. Thus our proposition of molecular
interaction theory bridges the missing link between the protochemical and biological
evolution.
Origin of chiral specificity
Another interesting feature of our proposition is that it gives a novel approach in
understanding the possible origin of chiral specificity (Balasubramanian, 1983). For
other explanations see proceedings of the 2nd International Symposium, 1981. The
specific nature of the hydrogen-bonding interaction between penta-ribonucleotides
and amino acids are such that L-amino acids interact more favourably (of the order of
5 kcal/mol) with β-D-ribonucleotides than D-amino acids. Thus there is a chiral
specificity between β-D-ribonucleotides and L-amino acids (Balasubramanian and
Seetharamulu, 1984). Thus in the possible origin of this genetic decoding apparatus, the
co-existence or conjunction of L-peptides and β-D-ribonucleotides becomes a necessity
because of their cooperative template fitting interaction; of course, the mirrored
combination of optical antipodes could also trigger the ordered synthesis of D-peptides
(figure 7).
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Balasubramanian
Figure 7. Schematic diagram of primitive tRNAs of opposite chiralities synthesising L- and
D-peptides.
In this context an interesting possibility suggests itself. Two protein synthesizing
machineries, one manufacturing L-peptides (say, L-system) and another D-peptides
(say, D-system) might have developed autonomously. The two systems are capable of
evolving independently. Natural mutations on the sequences of PIMs and the errors in
the primitive translation would bring in new sequences of polypeptides and the
Darwinian process of survival of the fittest would lead to more and more sophisticated
decoding systems. But L-system and D-system would take entirely different pathways
since each of them is dependent respectively on its own enentiomorphic PIM-sequences
Nucleotide-amino acid interactions
833
and the chance-mutations on them. At one stage, let us suppose that L-system develops
an enzyme that is capable of efficiently cleaving all D-peptides. Thus L-system attains
an evolutionary supremacy over D-system which would not be able to survive because
of this “killer enzyme” (figure 8). D-system could also develop a “killer enzyme” to Lsystem. But since the two systems are independently evolving, the probability of both
the systems developing a killer enzyme at the same time is extremely small and
whichever system happened to bring forth a killer enzyme for the other system,
survived to outlast the other in the evolutionary race.
Figure 8. Schematic diagram showing the dependence of polypeptide sequences on the PIM
sequence of the corresponding chirality. Sequences of L-enzyme and those of D-enzyme are
completely different. One of them could have first developed a killer enzyme for the other and
hence its evolutionary survival while the other could have been eliminated from our biosphere
even at the early stages of evolution.
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Balasubramanian
Since this hypothesis is based on a mechanism of active and effective destruction of
the system of opposite chirality this turns out to be a specific and forceful rationale for
the establishment of biological organisms of a given chiral specificity in a biosphere. It is
also testable in the sense that one could look for such an enzyme in the primitive or
archaic organisms. As such, the existence of antibiotics for the presently existing Lsystem gives credence to the hypothesis and it should be noted that peptide antibiotics
indeed contain D-amino acids that are inevitable for their function (Davies, 1977).
Conclusion
In conclusion we would like to bring out the fact that this postulate of molecular
interaction between pentanucleotides and amino acids possess almost all the salient and
unique features of the contemporary protein synthesizing machinery. They can be
summarized thus:
(i)
(ii)
(iii)
(iv)
(v)
(vi)
(vii)
(viii)
(ix)
(x)
(xi)
The rationale behind the origin of the uniquely peculiar adaptor strategy for
protein synthesis in biological processes is straightaway explained as the direct
consequence of this type of molecular interaction,
The triplet coding is a necessary outcome of the U-turn conformation of PIT as
elaborated earlier.
Again the wobble behaviour of the first base of PAC has been shown to be a
necessary consequence of the formation of U-turn hydrogen bond.
The glaring feature of the genetic code, viz, the middle base of codon playing a
key role, the first less so, and the third being almost non-specific is reflected in
primitive decoding system as the amino acid side chain neatly positioned near
the middle base of anticodon, is farther from the third base and farthest from
the first.
In our postulate, the possibility of dynamic interaction is such that the adaptor
mediated synthesis would proceed from 5'- to 3'-end of the mRNA, which is
what is happening in contemporary protein synthesis.
Similarly PIT’s hold on amino acid is such that the synthesis would proceed
from amino to carboxyl end for the polypeptide.
As discussed earlier PIT’s cleft is such that the non-proteinous amino acids
might have been eliminated from this system of polypeptide synthesis in the
early stages of evolution.
The remarkable features of stereospecificity between β-D-ribonucleotides and
L-amino acids and the possible emergence of chiral uniqueness in living
process has already been elaborated.
In PIT, uracil residue preceeding PAC plays a crucial role. In contemporary
tRNAs U is an invariant residue preceeding anticodon triplets.
In PIT, a purine succeeding PAC plays an important role in holding and
positioning an amino acid. And in present-day tRNAs the base that follows
anticodons is always a purine.
Last but not the least, crystal structures of tRNAs reveal the existance of Uturn conformation in the anticodon loop and it is perhaps the ‘fossil’ evidence
Nucleotide-amino acid interactions
835
for our molecular interaction theory for origin of life. It seems as though the
bits of a jig-saw puzzle fall into their right places when we consider this
particular molecular interaction between pentanucleotides and amino acids.
Acknowledgements
The author thanks Drs. P. Seetharamulu and G. Raghunathan for some of the
calculations carried out by them. A part of the work is supported by a grant from the
Department of Science and Technology, New Delhi, for “Studies on Origin and
Evolution of Life”.
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