The thermodynamic basis of the genetic code
A systematic investigation of the helixcoil transition of DNA’s as a function of sequence composition has lead t o the conclusion that the Gibbs energy per base pair is a zero-order property. There is a correlation between the observed codon usage frequency and the codonlanti-codon interaction Gibbs energy per triplet. We will show that the bias of codon usage reflects strategies for efficiency and accuracy in gene expression. Organization beyond the codon triplets is discussed. THE TWO KINGDOMS: THERMODYNAMICS AND GENETICS The great abstract value of thermodynamics stems from the fact that since the change of a system from its initial state to the final state does not depend on the path taken, we do not need to know anything about the mechanism. The great practical use of thermodynamics originates from the fact that changes in state functions such as energy or entropy do not depend on a particular path taken from one state to another, and therefore we will always be able to equate the end results of proceeding from one state (1) to another by two different paths. We can thereby obtain information about reactions that we could not study directly, such as those taking place within living cells. Specifically, thermodynamics gives a quantitative basis for predicting energy changes resulting from chemical reactions or accompanying conformational changes. This information is crucial in determining the reaction routes involved in various metabolic pathways. The full formal elegance of thermodynamics becomes evident from a careful development of the first and second laws, which is outside the scope of this discussion. However, there is a practical desire for thermodynamic data because they define the nature of the interand intra-molecular forces that stabilize a particular conformation of a biopolymer (2-4) . This interest intensified when it was realized that such data can serve as the basis for predicting sequence-specific secondary structural domains along linear polymer chains, such as DNA. DNA is the main creature in the other Kingdom, genetics. The formal elegance of thermodynamics is completely matched by that of the concept of the genetic code (5). After Avery’s demonstration that DNA is the genetic material, Chargaff’s analyses showing that the relative ratios of the four bases in genomic DNA were far from random, and Watson and Crick’s elegant demonstration of the 1357 Unauthenticated Download Date | 9/27/18 5:01 PM 1358 H. H. KLUMP AND D. L. MAEDER structure of the double helical conformation, it was only a matter of time before it was proposed that groups of three nucleotides (triplets) could serve the purpose of coding for the twenty amino acids found in almost all protein sequences (6). The code must be degenerate because the 64 permutations provide much more code words than needed for a unique correlation of a triplet and a specific amino acid. The experimental breakthrough in identifying which triplet determines which specific amino acid in a sequence came when Matthei and Nirenberg demonstrated that the synthetic messenger RNA poly-uridylic acid codes for the monotonous polypeptide poly-phenylalanine ( 7 ) . Use of increasingly more complex polynucleotides as synthesized in Khorana’s laboratory finally established the complete set of codons. It turns out that 61 out of 64 possible triplets correspond to amino acids, most of them being coded by more than one, and the residual three codons being stop signals. Consequently the smallest unit of the DNA sequence which is relevant in the genetic context is the triplet and not the single nucleotide. In non-coding regions of the total genome, other unit sizes may be considered (8). The aim of our approach is to combine the two Kingdoms in such a way that we can assign a characteristic thermodynamic parameter to any triplet. The parameter chosen is the interaction Gibbs energy between each codon and its anti-codon. In the following we will demonstrate that there is indeed a correlation between interaction Gibbs energy and the preference for certain codons in certain genes ( 9 ) . EXPERIMENTAL APPROACH TO THE INTERACTION GIBBS ENERGY The concerted thermal denaturation of a long DNA sequence, normally termed helix coil transition, is a sequence-dependent process. The H-bonds in the plane of the individual base pairs have to be broken; a process similar to the destabilization of H-bonds in melting ice. Accordingly, the temperature at which 50% of the bases are unpaired is called the melting temperature (Tm)(lO). The melting of DNA can conveniently be followed by recording the change in absorbance at 260 nm due to the unstacking of consecutive bases in the single strands or of partly overlapping bases in opposite strands. Stacking interactions contribute to the overall heli l t stability as much as the intact H-bonds (11). The helical structure is destabilized by electrostatic interactions between the backbone phosphate groups as well as by the conformat.iotia1 entropy decrease resulting from the freezing of free rotation around single bonds along the two backbones. The entropy term is expected to be sequence-independent. Since no bonds inside a particular base are involved, it is dependent only on the total number of degrees of freedom restricted upon helix formation. Using Boltzmann’s formalism (12) S = n.R.ln(w) to calculate the entropy change per subunit (nucleotide) the maximal entropy change should amount to 13 e.u. because in our case we have to consider six single bonds along the DNA backbone (n-6) and three preferred conformations per bond (w-3). As will be shown later, the experimental results fit very well with the above assumption (Fig. lc). Unauthenticated Download Date | 9/27/18 5:01 PM The thermodynamic basis of the genetic code 1359